Compounds that inhibit complement and/or suppress immune activity

ABSTRACT

The present invention is directed to compounds which suppress immune responses and/or selectively inhibit complement. These compounds contain an aromatic ring and are substituted dihydrobenzofurans, spirobenzofuran-2(3H)-cycloalkanes, and their open chain intermediates. The compounds of the present invention, and the pharmaceutically acceptable salts thereof, interrupt the proteolytic processing of C5 to bioactive components, exhibit immunosuppressive activities, and have therapeutic utility in the amelioration of disease and disorders mediated by complement and/or immune activity.

This application is a 371 of PCT/US/9109303 filed Dec. 6, 1991 which isa continuation-in-part of applcation Ser. No. 07/623,849 filed Dec. 6,1990, now U.S. Pat. No. 5,366,986, which is a continuation-in-part ofapplication Ser. No. 07/182,275 filed Apr. 15, 1988, now U.S. Pat. No.5,173,499.

1. FIELD OF THE INVENTION

The present invention relates to compounds which inhibit complementand/or possess immunosuppressive activity. In particular, the compoundsof the present invention, and the pharmaceutically acceptable saltsthereof, selectively inhibit complement at the C5 step of complementactivation. The compounds of the invention are substituteddihydrobenzofurans, spirobenzofuran-2(3H)-cycloalkanes, and their openchain intermediates that exhibit such inhibitory activates. Compounds ofthe invention also include 6,7-disubstitutedspirobenzofuran-2(3H)-cycloalkanes and 4-substitutedspirobenzofuran-2(3H)-cycloalkanes. The invention relates to the use ofthese compounds for therapy of immune and/or inflammatory disorders.

2. BACKGROUND OF THE INVENTION 2.1 The Complement System

The complement system is a group of proteins that constitutes about 10percent of the globulins in the normal serum of humans (Hood, L.E. etal. 1984, Immunology, 2d Edition, The Benjamin/Cummings Publishing Co.,Menlo Park, Calif., p. 339). Complement (C) plays an important role inthe mediation of immune and allergic reactions (Rapp, H. J. and Borsos,T., 1970, Molecular Basis of Complement Action, Appleton-Century-Crofts(Meredith), New York). The activation of C components leads to thegeneration of a group of factors, including chemotactic peptides thatmediate the inflammation associated with complement-dependent diseases.The sequential activation of the complement cascade may occur via theclassical pathway involving antigen-antibody complexes, or by analternative pathway which involves the recognition of certain cell wallpolysaccharides. The activities mediated by activated complementproteins include lysis of target cells, chemotaxis, opsonization,stimulation of vascular and other smooth muscle cells, degranulation ofmast cells, increased permeability of small blood vessels, directedmigration of leukocytes, and activation of B lymphocytes, macrophagesand neutrophils (Eisen, H.N., 1974, Immunology, Harper & Row,Publishers, Inc., Hagerstown, Md., p. 512).

During proteolytic cascade steps, biologically active peptide fragments,the anaphylatoxins C3a, C4a, and C5a (See WHO Scientific Group, WHOTech. Rep. Ser. 1977, 606, 5 and references cited therein), are releasedfromthe third (C3), fourth (C4), and fifth (C5) native complementcomponents (Hugli, T. E. CRC Crit. Rev. Immunol. 1981, 1, 321; Bult, H.and Herman, A. G. Agents Actions 1983, 13, 405). The C5a fragment, acationic peptide derived from the first 74 amino acids of theamino-terminus of the C5 alpha subunit (Tack, B.F. et al. Biochemistry1979, 18, 1490), is of particular pathological relevance. Regulation ofC5a activity is by the endogenous plasma enzyme carboxypeptidase N (E.C.3.4.12.7), which rapidly removes the carboxy-terminal arginine from C5a,producing the less potent but still active C5a des Arg. Reported effectsof C3a and C5a upon specific immune responses are listed in Table I.

                  TABLE I                                                         ______________________________________                                        EFFECTS OF COMPLEMENT COMPONENTS                                              C3a AND C5a ON SPECIFIC IMMUNE RESPONSES                                      Immune Response C3a        C5a/C5a des Arg                                    ______________________________________                                        Specific antibody production                                                                  Suppression                                                                              Enhancement                                        in response to sheep red                                                      blood cells                                                                   Polyclonal antibody                                                                           Suppression                                                                              Enhancement                                        production in response                                                        to Fc antibody fragment                                                       T cell proliferation                                                                          Suppression                                                                              Enhancement                                        in response to tetanous                                                       toxoid                                                                        T cell proliferation in                                                                       No effect  Enhancement                                        mixed lymphocyte reaction                                                     T cell-mediated cytotoxicity                                                                  Suppression                                                                              Enhancement                                        ______________________________________                                    

Among the wide variety of biological activities exhibited by C5a arecontraction of smooth muscle (Wissler, J. H. Eur. J. Immunol. 1972, 2,73), degranulation of mast cells (Johnson, A. R. et al., Immunol. 1975,28, 1067), secretion of azurophilic granular enzymes frompolymorphonuclear neutrophils (PMN) (Webster, R. O. et al.,Immunopharmacol. 1980, 2, 201), and the chemotaxis of PMN (Wissler, J.H. Eur. J. Immunol. 1972, 2, 73; Becker, E. L. Trends Pharmacol. Sci.1983, 4, 223) (Table II).

                  TABLE II                                                        ______________________________________                                        BIOLOGICAL EFFECTS OF C5a                                                     ______________________________________                                        I.        Stimulation of neutrophil functions                                           involved in inflammation                                                      A. chemotaxis                                                                 B. chemokinesis                                                               C. aggregation                                                                D. lysosomal enzyme release                                                   E. generation of toxic oxygen products                              II.       Smooth muscle effects                                                         A. stomach smooth muscle contraction                                          B. vasodilation                                                     II.       Promotion of histamine release                                                A. mast cells                                                                 B. basophils                                                        IV.       Immunoregulatory effects                                            ______________________________________                                    

The active chemotactic factor in vivo is considered to be C5a des Arg(Becker, E.L. Trends Pharmacol. Sci. 1983, 4, 223).

The C5a or C5a des Arg fragments have been implicated in theinfiltration of PMN (the chemotactic effect) in rheumatoid arthritis,certain forms of glomerulonephritis, experimental vasculitides such asthe Arthus reaction, the acute pneumonitis produced by the instillationof chemotactic factors into the lungs of experimental animals withresulting release of leukotrienes C-4 and D-4 (LTC₄ and LTD₄), etc. Inaddition, the interactions between C5a and neutrophils have beenconsidered to underlie tissue damage in several clinical situations. Forinstance, there exists a growing body of evidence for the role ofoxygen-derived free radicals in mediating myocardial tissue injuryduring myocardial ischemia and, in particular, during the phase ofmyocardial reoxygenation and reperfusion. Among a number of possiblesources of these radicals, the polymorphonuclear neutrophil has been thefocus of primary attention. Studies have documented that neutrophildepletion or suppression of neutrophil function results in a significantsalvage of myocardial tissue that is subjected to a period of regionalischemia followed by reperfusion (Simpson, P. J. and Lucchesi, B. R. J.Lab. Clin. Med. 1987, 110(1), 13-30). Neutrophil depletion in dogsresulted in significantly smaller myocardial infarcts after 90 minuteocclusion with 24 hour reperfusion (Jolly, S. R. et al. Am. Heart J.1986, 112, 682-690).

One study documented the activation of complement and generation ofoxygen-derived free radicals during cardiopulmonary bypass. Theadministration of protamine during cardiopulmonary bypass furtheractivated complement (Cavarocchi, N. C. et al., Circulation 1986, 74,130-133; Kirklen, J. K. et al. J. Thorac. Cardiovasc. Surg. 1983, 86,845-857). Also, recombinant tissue plasminogen activator (r-TPA), whichin recent clinical trials has been found to be an effective thrombolyticagent in patients with acute myocardial infarction, was shown toactivate complement. A striking increase in the level of C4a, C3a, andC5a was found in patients receiving r-TPA as compared to the level ofthese complement peptides before administration of the drug (Bennett, W.R. et al. J. Am. Coll. Cardiol. 1987, 10(3), 627-632). Schafer andco-workers were able to positively identify the deposition of terminalC5b-9 complement complex in myocardial cells located within zones ofinfarction in human tissue (J. Immunol. 1986, 137(6), 1945-1949).Likewise, the selective accumulation of the first component ofcomplement and leukocytes in ischemic canine heart muscle has been found(Rosen, R. D. et al. Circ. Research, 1985, 57, 119-230). In one study,the depletion of complement was found to increase the blood flow inischemic canine myocardium. This increased blood flow was found, inturn, to increase the supply and utilization of oxygen in complementdepleted animals versus control animals (Grover, G. J. and Weiss, H. R.Basic Res. Cardio. 1987, 82(1), 57-65). Complement activation is alsobelieved to initiate adult respiratory distress syndrome (ARDS). Thissyndrome, also known as adult respiratory failure, shock lung, diffusealveolar damage, or traumatic wet lungs, is characterized clinically bythe rapid onset of severe life-threatening respiratory insufficiencythat is refractory to oxygen therapy. (Miescher, P. A. andMuller-Eberhard, H. J., eds., 1976, Text Book of Immunopathology, 2dEd., Vols. I and II, Grune and Stratton, New York; Sandberg, A. L.,1981, in Cellular Functions in Immunity and Inflammation, Oppenheim, J.J. et al., eds. Elsevier/North Holland, N.Y., p. 373; Conrow, R.B. etal. J. Med. Chem 1980, 23, 242; Regal, J. F.; and Pickering, R. H. Int.J. Immunopharmacol. 1983, 104, 617). Some of the clinical implicationsof C5a release are listed in Table III.

                  TABLE III                                                       ______________________________________                                        CLINICAL IMPLICATIONS OF C5a RELEASE                                          ______________________________________                                        Rheumatoid Arthritis                                                            Acute Gouty Arthritis                                                         Acute Immunological Arthritis                                               Pulmonary Disorders                                                             Adult Respiratory Distress Syndrome                                           Pulmonary Dysfunction - Hemodialysis                                          Chronic Progressive Pulmonary Dis-Cystic Fibrosis                             Byssinosis                                                                    Asbestos-Induced Inflammation                                               Inflammation of Systemic Lupus Erythematosus                                  Inflammation of Glomerulonephritis                                            Purtscher's Retinopathy                                                       Hemorrhagic Pancreatitis                                                      Renal Cortical Necrosis                                                       Primary Biliary Cirrhosis Inflammation                                        Nephropathology                                                               Cranial Nerve Damage in Meningitis                                            Tumor Cell Metastasis                                                         Extended Tissue Destruction in Myocardial Infarction                          Extended Tissue Destruction in Burns                                          ______________________________________                                    

2.2. CELL-MEDIATED IMMUNE RESPONSES

A variety of immune responses independent of the complement system areknown to be mediated by specifically reactive lymphocytes. Theseresponses may give rise to autoimmune diseases, hypersensitivity, orsimply allergic reactions. Some examples of these responses includedelayed-type hypersensitivity, allograft rejection, graft versus hostdisease, drug allergies, or resistance to infection. Autoimmunedisorders may include atrophic gastrititis, thyroiditis, allergicencephalomyelitis, gastric mucosa, thyrotoxicosis, autoimmune hemolyticanemia, and sympathetic ophthalmia (Eisen, H. N., 1979, Immunology,Harper and Row, Hagerstown, Md., pp. 557-595).

2.3. ORGANIC COMPOUNDS WHICH INHIBIT COMPLEMENT, AMELIORATEINFLAMMATION, AND/OR POSSESS IMMUNOSUPPRESSIVE ACTIVITY

Many chemicals have been reported to diminish complement-mediatedactivity. Such compounds include: amino acids (Takada, Y. et al.Immunology 1978, 34, 509); phosphonate esters (Becker, L. Biochem.Biophy. Acta 1967, 147, 289); polyanionic substances (Conrow, R. B. etal. J. Med. Chem. 1980, 23, 242); sulfonyl fluorides (Hansch, C.;Yoshimoto, M. J. Med. Chem. 1974, 17, 1160, and references citedtherein); polynucleotides (DeClercq, P. F. et al. Biochem. Biophys. Res.Commun. 1975, 67, 255); pimaric acids (Glovsky, M. M. et al. J. Immunol.1969, 102, 1); porphines (Lapidus, M. and Tomasco, J. Immunopharmacol.1981, 3, 137); several antiinflammatories (Burge, J. J. et al. J.Immunol. 1978, 120, 1625); phenols (Muller-Eberhard, H. J. 1978, inMolecular Basis of Biological Degradative Processes, Berlin, R. D. etal., eds. Academic Press, New York, p. 65); and benzamidines (Vogt, W.et al Immunology 1979, 36, 138). Some of these agents express theiractivity by general inhibition of proteases and esterases. Others arenot specific to any particular intermediate step in the complementpathway, but, rather, inhibit more than one step of complementactivation. Examples of the latter compounds include the benzamidines,which block C1, C4 and C5 utilization (Vogt, W. et al. Immunol. 1979,36, 138).

2.4. SPIROBENZOFURAN-2(3H)-CYCLOALKANES AND K-76

K-76 is a fungal metabolite from Stachybotrys complementi nov. sp. K-76.Metabolite K-76 has a drimane skeleton combined with a benzene ringattached through a spirofuran, and has been determined as6,7-diformyl-3', 4', 4a', 5', 6', 7', 8', 8a'-octahydro-4,6',7'-trihydroxy-2', 5', 5', 8a'-tetramethyl spiro[(1'(2'H)-naphthalene-2(3H)-benzofuran] (Kaise, H. et al. J. Chem. Soc.Chem. Commun. 1979, 726). The monocarboxylic acid derivative, K-76 COOH,is obtained when K-76 is selectively oxidized by silver oxide. (Corey,E. J. and Das, J. J. Amer. Chem. Soc. 1982, 104, 5551).

Both K-76 and K-76 COOH have been shown to inhibit complement mainly atthe C5 step (Hong, K. et al. J. Immunol. 1979, 122, 2418; Miyazaki, W.et al. Microbiol. Immunol. 1980, 24, 1091). In a classical hemolyticreaction system, hemolysis of sensitized sheep erythrocytes by guineapig serum was reduced 50% by K-76 at 7.45×10⁻⁵ M, or K-76 COONa at3.41×10⁻⁴ M (Hong, K. et al. J. Immunol. 1979, 122, 2418; Miyazaki, W.et al. Microbiol. Immunol. 1980, 24, 1091). Similar results wereobserved in a hemolytic reaction system via the alternative pathway ofcomplement activation.

Both K-76 and K-76 COOH prevented the generation of a chemotactic factorfrom normal human complement (Bumpers, H. and Baum, J. J. Lab. Clinc.Med. 1983, 102, 421). K-76 has been shown to reduce the amount ofprotein excreted in urine of rats with nephrotoxic glomerulonephritis(Iida, H., et al., Clin. Exp. Immunol. 1987, 67, 130-134), and isreported to greatly increase the survival of mice with a spontaneoussystemic lupus erythematosis-like disease and to suppress Forssman shockin guinea pigs and mice (Miyazaki, W. et al. Microbiol. Immunol. 1980,24, 1091). At high concentrations of K-76 or K-76 COOH, some inhibitionof the reactions of C2, C3, C6, C7, and C9 with their respectivepreceding intermediaries is exhibited. However, both compounds'inhibitory action is mainly the generation in vitro of EACl, 4b,2a,3b,5b(sensitized sheep erythrocytes carrying the indicated complementcomponents) from C5 and EACl,4b,2a,3b; the acceleration of the decay ofany EACl,4b,2a,3b,5b present; and blocking generation of the chemotacticpeptides (Hong, K. et al. J. Immunol. 1981, 127, 109; Ramm, L.E., et al.Mol. Immunol. 1983, 20, 155) .

K-76 COOH is also reported to be an anti-hepatitic agent (West GermanPatent Application, Publication No. 3,031,788, published Mar. 12, 1981,by Shinohara, M. et al.), and possesses the ability to inhibitantibody-dependent cell-mediated cytotoxicity and natural killer lyticactivity (Hudig, D. et al. J. Immunol. 1984, 133, 408-413). K-76 or K-76COOH has also been reported to inhibit the C3b inactivator system ofcomplement (Hong, K. et al. J. Immunol. 1981, 127, 104-108).Semi-synthetic derivatives of K-76 have been patented as anti-allergy,anti-tumor, and anti-nephritic agents (Belgium Patent No. 867,095,published Nov. 16, 1978, by Shinohara, M. et al.). The isolation ofK-76, its uses in the treatment of autoimmune diseases, and thepreparation of its derivatives have been described in a number ofpatents (See Japanese Patent Applications (Kokai), Publication Nos. 54092680 (published Jul. 23, 1979), 54 106458 (published Aug. 21, 1979),57 083281 (published May 25, 1982), by Shinohara, M. et al.; JapanesePatent (Kokoku) No. 85 030289 (published Mar. 20, 1979)).

A number of additional compounds which contain the substructure of aspirobenzofuran-2(3H)-cycloalkane are known. These compounds includegriseofulvin (Weinberg, E.D., 1981, in Principles of MedicinalChemistry, 2d Ed., Foye, W. O., ed., Lea & Febiger, Philadelphia, Pa.,p. 813), isopannarin (Djura, P. and Sargent, M. V. Aust. J. Chem. 1983,36, 1057), and metabolites of Siphonodictyon coralli-phagum (Sullivan,B., et al. Tetrahedron 1981, 37, 979).

The general synthetic methodology utilized for the synthesis of thecompounds of the present invention involves regioselective aromaticlithiation reactions. Numerous oxygen-containing heterocycles have beenmade using regioselective aromatic lithiation reactions, but fewpreparations of dihydrobenzo[b]furans have been described (Narasimhan,N. S. and Mali, R. S. Synthesis 1983, 957). Three. reported syntheses ofK-76 itself (Corey, E. J. and Das, J. J. J. Am. Chem. Soc. 1982, 104,5551; McMurray, J. E. et al. Am. Chem. Soc. 1985, 107, 2712 Mori, K. etal. Ann. Chem. 1988, 107-119) utilize metalation techniques in thecoupling of the terpenoid portion to the aromatic moiety, but neitherprovide sufficient flexibility to allow for analog preparations viasubsequent elaboration of the aromatic ring.

3. SUMMARY OF THE INVENTION

The present invention is directed to compounds which suppress immuneresponses and/or selectively inhibit complement. In a specificembodiment, such compounds interrupt the proteolytic processing of C5 tobioactive components, blocking the release of C5a. The compounds of thepresent invention also exhibit immunosuppressive activities, such as forexample, the ability to inhibit natural killer activity, lymphocyteproliferations, and T cell activation. The compounds of the presentinvention have therapeutic utility in the amelioration of disease anddisorders mediated by complement and/or immune activity. In specificembodiments, they may be used for the treatment of autoimmune disease orthe many diseases associated with the "inappropriate" activation of thecomplement system. In specific embodiments, a compound of the inventionhas greater activity than the natural product K76.

The invention further provides improved synthetic routes for thepreparation of the compounds.

In one embodiment of the present invention, compounds are provided whichhave selectivity in inhibition of C5a release. In particular, suchcompounds can have utility in limiting the extent of trauma-inducedtissue destruction, in the prevention and/or treatment of adultrespiratory distress syndrome and damage induced by ischemic heartconditions.

In other specific embodiments, compounds of the invention which exhibitimmunosuppressive activity can be used in the prevention and/ortreatment of autoimmune disease or the rejection of transplanted organsand/or tissues.

A further embodiment of this invention includes the combined therapythat can be obtained by treating patients with disorders that areroutinely treated with thrombolytic agents such as tissue plasminogenactivator, streptokinase or urokinase (e.g. myocardial infarctionpatients) with a combination of the compounds of this invention and theroutinely administered thrombolytic compounds.

The present invention is also directed to pharmaceutical compositionscomprising such compounds or the salts thereof.

3.1 DEFINITIONS

As used herein, the following abbreviations and terms shall have themeanings indicated:

n-BuLi=n-butyllithium

t-BuLi=tert-butyllithium

t-BuSLi =lithium tert-butylthiolate

c=complement

CHO=Chinese hamster ovary

CPM=counts per minute

Et₂ O=diethyl ether

HMPA=hexamethylphosphoric triamide

IgG=immunoglobulin G

^(i) PrOH=isopropanol

IR=infrared

K-76 COOH=the monocarboxylic acid derivative of K-76

K-76 COONa=the sodium salt of the monocarboxylic acid derivative of K-76

LAH=lithium aluminum hydride

MOM=methoxymethyl group

NK=natural killer

NMR=nuclear magnetic resonance

PBL=peripheral blood lymphocyte(s)

PCC=pyridinium chlorochromate

PHA=phytohemagglutinin

PMN=polymorphonuclear cells

RLi=alkyllithium

THF=tetrahydrofuran

TLC=thin layer chromatography

TMEDA=N,N,N',N'-tetramethyl-ethylenediamine

TMS=tetramethylsilane

TriMEDA=N,N,N' trimethylethylenediamine

The term "bioisosteric" group, as used in the present invention,describes an alternative chemical group whose electronic configurationis substantially analogous with the group to be replaced such that thepolarity and charge of the whole molecule do not change. However,variations in the size, number of atoms or electron structure of thebioisosteric group (or "bioisostere") are permitted which variations mayaffect its function. Bioisosteres may be acidic (e.g., capable ofreleasing a proton and, subsequently, bearing a negative charge), basic(e.g., capable of being protonated and, subsequently, bearing a positivecharge) or neutral (e.g., not normally capable of functioning as anacidic or basic group).

Unless otherwise stated or indicated, the term "alkyl" as used hereinrefers to methyl, ethyl, and n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, or tert-butyl groups. The term "alkanol" denotes a compoundderived from coupling an alkyl group and hydroxyl radical. Similarly,the term "alkoxy" refers to methoxy, ethoxy, n-propyloxy, isopropyloxy,n-, iso-, sec, and tert-butoxy, phenoxy, benzyloxy groups andsubstituted derivatives thereof. The term "lower" refers to thenumerical range of 1 to 4 carbon atoms and includes linear or branchedskeletons.

Unless otherwise stated or indicated, the term "halogen" as used hereinincludes fluorine, chlorine, bromine, and iodine.

Unless otherwise stated or indicated, a given structure, formula, ornomenclature for the substituted dihydrobenzofuran analogs of thisinvention shall subsume all stereoisomers thereof.

Unless otherwise stated or indicated, a reference made to a finalcompound of the invention which is a carboxylic acid, is also meant toinclude the salt form of such carboxylic acid such as alkali andalkaline-earth metal salts obtained therefrom.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the infrared spectrum of compound 11a. The spectrumis presented for the compound pelletized in potassium bromide.

FIG. 2 demonstrates the inhibition of complement peptide C5a or C3aproduction by compound 11a. The inhibition (as described in Section 6.6,infra) of C5a and C3a production is shown as a function of compound 11aconcentration.

FIG. 3 demonstrates the inhibition of complement-mediated hemolysis bycompound 11a. Inhibition of complement-mediated hemolysis, assayed asdescribed in Section 6.6.2 infra, is shown as a function of compound 11aconcentration.

FIG. 4 demonstrates the inhibition of proliferation of peripheral bloodlymphocytes (PBL) by compound 11a. ³ H-Thymidine incorporation byPHA-stimulated PBL is shown as a function of compound 11a concentration.

FIG. 5 demonstrates the inhibition of proliferation of PBL by compound11a. ³ H-Thymidine incorporation by antiCD3 antibody-stimulated PBL isshown as a function of compound 11a concentration.

FIG. 6 demonstrates the inhibition of interleukin-2 receptor (IL-2R)release from PBL by compound 11a. The level of IL-2R in the supernatantof PBL cultures stimulated with anti-CD3 antibody, in the presence ofcompound 11a, was measured by use of an enzyme-linked immunosorbentassay, and is shown as a function of compound 11a concentration.

FIG. 7 shows the inhibition of CD8 protein release from PBL by compound11a. The level of CD8 in the supernatant of PBL cultures stimulated withanti-CD8 antibody, in the presence of compound 11a, was measured by useof an enzyme-linked immunosorbent assay, and is shown as a function ofcompound 11a concentration.

FIG. 8 demonstrates the inhibition of complement-mediated hemolysis bythe disubstituted spirobenzofuran compounds 62, 66, and 68. Inhibitionis shown as a function of compound concentration .

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds which inhibit complementand/or possess immunosuppressive activity. The compounds of theinvention contain an aromatic ring and are substituteddihydrobenzofurans, spirobenzonfuran-2(3H)-cycolakanes, and their openchain intermediates. In particular, such compounds can be partialanalogs of the fungal metabolite K-76.

The complement inhibitors of the invention inhibit C5 activation, thatis, the proteolytic generation of bioactive complement fragments C5a andC5b from C5. Such compounds have value in the treatment or prevention ofdiseases or disorders associated with undesirable or inappropriateactivation of the complement system. In specific embodiments, thecompounds of the invention can be used in the treatment of inflammatorydisorders. They may also be used for the treatment of cardiovasculardisease.

The present invention also relates to compounds which possessimmunosuppressive activity. In particular, such compounds inhibit immuneresponses. In specific embodiments, the compounds of the invention caninhibit the killing activity of mononuclear cells, lymphocyteproliferation and/or activation. The immunosuppressive compounds of theinvention can be valuable in the treatment of various immune disorders.

Furthermore, the compounds of the invention may possess one or more ofthe K-76-1ike activities described supra in Section 2.4 and in thereferences cited therein.

The compounds of the present invention, and the intermediates andmethods used in their preparation, are described in detail below.

5.1. COMPOUNDS WHICH INHIBIT COMPLEMENT AND/OR SUPPRESS IMMUNE ACTIVITY

The present invention relates to organic compounds which inhibitcomplement and/or suppress immune activity. The organic compounds of theinvention comprise substituted dihydrobenzofurans of the general formula3 and substituted spirobenzofuran-2(3H)-cycloalkanes of the generalformula 4. The groups represented by R and R₁ -R₄ include, among others,hydrogen and linear or branched lower alkyl groups having 1 to 4 carbonatoms as defined previously in Section 3.1, supra. In addition, R₁ -R₄may each ##STR1## independently represent halogen, amino, amidic,hydroxyl, hydroxyalkyl, alkyloxy, nitro, formyl, acetal, carboxylicacid, trifluoroacetyl, N-substituted lower alkyl carbamoyl, substitutedvinyl having up to 10 carbon atoms, an alkylidene group having up to 20carbon atoms, an aliphatic acyl, a substituted aliphatic acyl, anaromatic acyl, a substituted aromatic acyl, a sulfamoyl, an aminomethyl,N-(lower alkyl)aminomethyl, a N, N-di(lower alkyl) aminomethyl, aheterocyclic ring bearing at least one heteroatom selected fromnitrogen, oxygen or sulfur (e.g., a tetrazole or oxazoline group), anN-acylcarbamoyl, an amidino or a hydrazide. Moreover, R₁, R₂ and thecarbon atoms to which they are attached may together form a five-orsix-membered ring (e.g., a cyclic anhydride, such as a phthalicanyhydride derivative; a lactone; or a hydroxy-substituted lactone).

Other groups which may be represented independently by R₃ and R₄ includehydrocarbons of 4 to 24 carbon atoms which may be of medium-length,long-chain, linear, branched, cyclic, saturated, unsaturated,unsubstituted, or heteroatom substituted. Moreover R₃, R₄, and thecarbon atom to which they are attached may form a cyclic hydrocarbongroup of 5-24 carbon atoms which may include a five-, six-, orseven-membered saturated or unsaturated ring comprised exclusively ofcarbon and hydrogen, or in combination with a heteroatom. The ring maybe unsubstituted or may contain extra-cyclic heteroatom or hydrocarbonsubstituents.

This invention also relates to synthetic open chain intermediatecompounds of the general formula S wherein R, R₁, and R₂ are defined asabove for formulae 3 and 4. In addition, R₅ represents hydrogen, loweralkyl groups, or suitable hydroxyl protecting groups such asmethoxymethyl, tetrahydropyranyl, 2-methoxypropyl,2-methoxyethoxymethyl, triarylmethyl, benzyl, methylthiomethyl, ortert-butyldimethylsilyl group. The R₆ group encompasses chemical groupsrepresented by R₁ -R₄ as defined above for formulae 3 and 4 as well assubstituent cyclohexenylmethyl (5a), limonenyl (5b), and carvone-deriveddiol acetonide (5c) groups. Other compounds may also be derived from##STR2## intermediates 5 and 5a-c, which are, in turn, converted toproducts of general formulae 3 or 4 as discussed in the followingsections. Table V lists representative compounds which comprise thegeneral formula 4 of the present invention; this list is not intended tobe comprehensive.

                  TABLE V                                                         ______________________________________                                        COMPLEMENT INHIBITORS OF THE                                                  PRESENT INVENTION.sup.a                                                       ______________________________________                                         ##STR3##                                                                                   ##STR4##                                                                               ##STR5##                                                                            ##STR6##                                         ______________________________________                                         .sup.a Substituents on the spirocyclohexane ring may also be present;         e.g., 10isopropenyl.                                                     

The substituted dihydrobenzofuran and spiro-benzofuran-2(3H)-cyclohexanecompounds of the present invention of the general formulae 3, 4, thesynthetic intermediates of the general formula 5, and the salts thereofexhibit complement inhibition, as manifested by inhibition ofcomplement-mediated C5a production and/or inhibition ofcomplement-mediated hemolysis. The complement-inhibitory properties ofthe compounds of the invention can be evaluated by modification of knowntechniques, e.g., the assay described in Section 6.6.1, infra.

Treatment of the compounds of the present invention with appropriatebasic reagents provides pharmaceutically acceptable salts thereof. Whena carboxylic acid group is present in the compounds of the invention, apharmaceutically acceptable ester can also be prepared by treatment witha suitable esterifying group under appropriate conditions.

5.2. SYNTHETIC PROCESSES

Processes are provided which comprise chemical steps for the synthesisof the compounds of the invention.

Such processes of the invention are diagrammed in detail in Scheme 1(See infra Section 5.2.2). The synthetic scheme depicted in Scheme 1provides a shorter and more flexible route than one based on thesyntheses of K-76 (Corey and Das J. Am. Chem. Soc. 1982, 104, 5551;McMurray et al. J. Am. Chem. Soc. 1985, 107, 2712). Retrosyntheticevaluation of all the final compounds produced according to Scheme 1ultimately results in two fragments; an aliphatic and an aromaticportion. These two portions can be joined by using regioselectiveortho-lithiation and subsequent alkylation. Two different strategies canthen be employed for the production of the final compound of theinvention: initial cyclization followed by aromatic functionalization orvice versa.

5.2.1. PREPARATION OF COMPOUNDS OF GENERAL FORMULA 5

The intermediates of the general formula 5 of the invention can beprepared by various methods depending on the types of substituentspresent therein.

The aliphatic substituent, R₆, of compounds of the general formula 5 arederived from suitable alkylating agents. For example,1-bromomethylcyclohexene, 6a, can be used as the starting material forthe preparation of compound 5a. Compound 6a can in turn be prepared fromcyclohexanone or directly from alkyl cyclohexenecarboxylate, accordingto a known precess (Wheeler, O. H. and Lerner, I. J. Am. Chem. Soc.1956, 78, 63; Adams, R. and Thai, A. F. Org. Synth. 1932, 1, 270;Lythgoe, B. et al. J. Chem. Soc. 1956, 406). In a particular example,cyclohexanone is converted to its cyanohydrin, then dehydrated to thecyanoalkene, followed by alcoholysis to an alkyl cycloalkenecarboxylate.Lithium aluminum hydride reduction of the ester followed by halogenationwith phosphorus trihalide provides compound 6a.

Limonenyl chloride, 6b, is obtained readily from limonenyl alcohol bythe action of triphenylphosphine in excess halogenated solvent. Thisconversion provides optically active allylic halide from opticallyactive limonenyl alcohol. A procedure analogous to that developed byCrawford and co-workers (J. Am. Chem. Soc. 1972, 94, 4298) is used toprepare optically active alcohol by the sequential lithiation ofcommercially available homochiral limonene, oxygenation, and reductionof the resultant hydroperoxide with aqueous sodium sulfite.

A third halointermediate, 6c, is derived from a multistep sequencestarting from optically active R-(-)or S-(+)-carvone. The steps of thesynthesis involve reduction of the e,β-unsaturated ketone to the allylicalcohol, epoxidation, reduction to the diol, acetonide formation, andtreatment of the unsaturated acetonide with calcium hypochlorite toyield the allylic chloride. The coupling reaction is generally carriedout immediately after purification of these unstable allylic halides.##STR7##

The aromatic segment of the compounds of the general formula 5 isobtained from substituted alkoxyphenols or resorcinols. For example,3-methoxymethoxyanisole, 7, is obtained from the reaction of3-methoxyphenol with chloromethyl methyl ether in a stirred suspensionof anhydrous potassium carbonate in acetonitrile (Rall, G. J. H. et al.Tet. Lett. 1976, 1033). Anhydrous conditions and the proper ratio ofsubstrate to solvent are critical to the success of this reaction. Forreactions employing quantities of reagents above 5 grams, a usefulalternative procedure uses the preformed sodium aryloxide, chloromethylmethyl ether, and dimethylformade as solvent (See, Rall, G.J.H. et al.supra). ##STR8##

The group R of formulae 5 and 5a-c should be stable to cleavage underthe conditions used to hydrolyze or remove the protecting group R₅ andthe subsequent cyclization of the resulting free phenol according tosteps 3 or 4 of Scheme 1. In addition, R₅ is preferably a potentiallychelating group which can promote the regioselective ortho-metalationrequired to introduce substituents R₂ and R₆ of formula 5. Compound 7embodies the preferred protecting groups for resorcinol (R=methyl andR=methoxymethyl). The methoxymethyl or MOM ethers can be easily removedin the presence of methyl ethers (Narasimhan, N. S. et al. Synthesis1979, 906), and the ortho directing power of the MOM group has beenshown to be greater than that of a methyl ether (Ronald R.C. Tet. Lett.1975, 3973).

In an alternative embodiment, the commercially available compound3,5-dimethoxybenzyl alcohol, 8, can be used as the aromatic segment forcoupling to the allylic halide (See Scheme 2, infra). ##STR9##

The efficacy of the coupling reaction between the metalated aromaticsegment and thealiphatic group is greatly influenced by a number offactors including the degree of aggregation of the metalating species,typically an alkyllithium reagent (Gschwend, H. W. and Rodriguez, H. R.Org. React. 1979 , 26, 1). The choice of solvent and any additives suchas N, N, N',N'-tetramethylethylenediamine (TMEDA) orhexamethylphosphoric triamide (HMPA), in turn, strongly influences thisaggregation state. Solvent(s), additive(s), temperature, reaction time,and reagent stoichiometries for coupling the respective substratesshould be optimized. Suitable solvents include anhydrous aprotic organicsolvents such as tetrahydrofuran (THF), diethyl ether (Et₂ O), dioxane,and diglyme. Surprisingly, we have discovered that dry hexane is apreferred solvent for selective metalation ortho to a chelating grouplike OMOM and lithioalkoxymethyl (kinetic conditions), while THF/TMEDAsolvent mixtures, generally, provide for metalation at the thermodynamicsite, between the two inductively electron-withdrawing methoxy groups in8, for example. In one particular embodiment, n-butyllithium (n-BuLi) inTHF/TMEDA or hexane/TMEDA allows for the coupling of aromatic segment 7with allylic bromide 6a; however, only THF/TMEDA allows coupling of 6awith aromatic segment 8 at the desired 4-position (thermodynamic). Whenthe coupling between 6a and 8 is carried out in hexane, lithiation andcoupling occurs in the 2-position of 8 (the kinetically favoredposition).

The aromatic substrate is dissolved in the solvent such as dry THF andthe like. The TMEDA is then added and is preferably approximatelyequimolar to the amount of alkyllithium used. In a preferred embodiment,the amount of alkyllithium (RLi) slowly added at 0° C. is approximately1.1 equivalents per equivalent of a compound of the type such as 7 andis approximately 2.2 equivalents per equivalent of a substrate such as 8that has an additional acidic hydrogen. Moreover, the addition ofapproximately 1.2 equivalents of a copper salt per equivalent of thelithiated aromatic substrate is preferred to obtain optimum yields fromthe coupling reaction. Most sources of copper(I) are suitable includingcuprous bromide dimethylsulfide, tetrakis (acetonitrile) copper(I)tetrafluoroborate, and the cuprous halides. The use of cuprous iodide ispreferred.

Careful control of temperature is important for the success of thearomatic alkylation. While the lithiation is carried out at zero degreeto ambient temperature, the subsequent reactions are carried out at lowtemperature. In a preferred example, the aryllithium reagent is cooledto -78° C. followed by the addition of copper(I) salt. The resultingmixture is subsequently stirred at -40° C., a temperature at which thecorresponding arylcuprate species can form and is relatively stable. Thecopper reagent is then recooled to -78° C. prior to the addition of theelectrophile. Carrying out the the addition at higher temperatures candiminish the yields of coupled products such as 5. It is important thatall manipulations discussed supra be performed in the absence of oxygenand moisture, and are preferably carried out under an inert atmospheresuch as dry nitrogen or argon.

The product mixture which is obtained from the coupling reaction canthen be worked up by washing several times with a moderately basicaqueous solution, e.g., saturated aqueous sodium bicarbonate (NaHCO₃).The organic phase can be dried by shaking with a drying agent such asmagnesium sulfate, filtered from any solids, and concentrated under amild vaccum to provide the crude product. Further purification may thenbe effected by procedures typically employed in the art (e,g.,fractional distillation, fractional crystallization, or chromatographicseparation).

5.2.2. PREPARATION OF COMPOUNDS OF THE GENERAL FORMULAE (3 AND 4)

Substituted dihydrobenzofuran derivatives of the general formula 3 withsubstituents OR and R₁ -R₄ as defined supra in Section 5.1 can beobtained by the metalation and subsequent alkylation of coupledintermediates such as those described in the preceding section, followedby hydrolysis of the protecting groups and cyclizaiton of theene-phenol. This synthetic pathway is illustrated in Scheme 1, infra.##STR10##

In a preferred embodiment, the protected resorcinol is lithiated andalkylated in step 1 of Scheme 1 using the allylic bromide 6a as theelectrophile. The resulting coupled product 9a can then be dissolved indry hexane and treated dropwise with alkyllithium in the presence ofTMEDA (step 2) at 0° C. Cooling the reaction mixture to -78° C. andaddition of the desired electrophile introduces the substituent at thisstage.

Generally, the MOM protecting group of intermediates such as 9, with orwithout the R₂ substituent, may be hydrolyzed by stirring the compoundin 5% hydrochloric acid (HCl)/ether mixtures overnight. Deprotection tothe free phenol may also be effected by a solvent mixture comprised of 4N HCl/^(i) PrOH, especially for carboxaldehyde containing derivatives,which need to be stirred for several days. The cyclization step can beachieved most conveniently by stirring the substrate in the presence ofAmberlyst resin in a non-polar solvent followed by filtration. In thisparticular process, the resin is washed with fresh solvent, and thefiltrates are combined and concentrated to give the desired crudecyclized product in good yield. In the particular case where R₂ =--COOHin intermediate 10a (Scheme 1, step 2, E⁺ =CO₂) the cyclization of thephenol (steps 3 and 4) can be achieved by stirring with Amberlyst resinin benzene for 24-72 hours at room temperature. Some difficulty isencountered in the cyclization of intermediates with hydroxymethylsubstituents (e.g., R₂ =--CH₂ OH). A different route is preferred forsynthesis of cyclized products with this substituent (see below).

As discussed supra, a variety of R₂ substituents may be introducedbefore the cyclization step. For example, addition of the followingelectrophiles to the aryllithium species produces the indicatedfunctional group (these examples serve only to illustrate a possibletechnique and are not meant to be limiting): carbon dioxide (carboxyl);ethyl chloroformate or diethyl carbonate (carbethoxy); dimethylformide(formyl); methylisocyanate (N-methylcarbamoyl); tert-butylisocyanate(N-tert-butylcarbamoyl); paraformaldehyde (hydroxymethyl);trifluoroacetic anhydride (trifluoroacetyl); bromine (bromide); chlorine(chloride); iodine (iodide); substituted vinyl iodides (substitutedvinyl group). Other compounds may, in turn, be obtained by modificationof these functional groups by known methods.

The cyclized methyl ether derivatives of the type, 11, can be convertedto the free phenols, 14 (Scheme 1, step 5), by known methods. Theseprocedures include but are not limited to treatment of the methyl etherwith boron trihalides (McOmie, J.F.W. and West, D. E. Org. Synth. 1973,5, 412; Grieco, P. A. et al. J. Org. Chem. 1975, 40 1450), borontrifluoride in the presence of thiols (Fujita, E. et al. Ibid. 1979, 44,1661; Fujita, E. J. Chem. Soc. Perkin Trans. 1 1976, 44, 4444), andlithium tert-butylthiolate salts (t-BuSLi) in hexamethylphosphorictriamide (McMurry, J. E. and Erion, M. D. J. Am. Chem. Soc. 1985, 107,2712). The use of t-BuSLi is preferred.

Open-chain coupled products such as 9b or 9c (See Scheme 1) give rise tocompounds such as 21 or 25, respectively. The latter intermediates,wherein the olefinic bond involved in the cyclization is exocyclic, orother derivatives containing simple unsaturated aliphatic groups,produce dihydrobenzofuran derivatives disubstituted at the 2-positionupon deprotection and treatment with Amberlyst ion-exchange resin (Seee.g., 20). ##STR11##

Slightly more complex analogs containing a diol group, such as 24 or 27,infra, can be obtained in this manner.

The use of 3,5-dimethoxybenzyl alcohol, 8, as the starting aromaticcomponent has the advantage of having the R₁ substituent already inplace. Compound 8 is dissolved in THF and is treated at 0° C. with 2.0equivalents of n-butyllithium in the presence of TMEDA (2.0 equiv). Thebenzyl alcohol is lithiated predominantly at the 4-position after about2 hours. Formation of the arylcuprate at -40° C. and addition of thealkylating agent completes the synthesis of 13 (Scheme 2, step 1) with asmall amount of the isomer 13' also being formed. Fractionalcrystallizaiton of the product mixture provides pure 13.

As previously alluded to, cyclization of intermediates withhydroxymethyl substituents can be problematic. In one embodiment, aremedy to this problem involves converting the benzyl alcohol 13c to theformyl compound 13b (See Scheme 2, step 2). This procedure can beaccomplished by a Swern oxidation or, more preferrably, by usingpyridinium chlorochromate in "buffered" methylene chloride. One of thesymmetrical methyl ethers is then selectively cleaved (step 3) andcyclized in the normal fashion to yield the 6-substituted formyl analog30b. Oxidation of the formyl group to the carboxylic acid (e.g., 30a or31a) can be effected by a number of reagents either before or after thecyclization of step 4. Suitable inorganic oxidizing agents include butare not restricted to permanganate salts, manganese oxide, chromic acid,chromate salts, silver oxide, silver nitrate, nickel peroxide, andcerium salts such as cerium sulfate, cerium oxide and ceriumperchlorate. Silver-oxide is preferred. The benzyl alcohol, 30c (4,R=CH₃, R₁ =CH₂ OH, and R₂ =H), is obtained by reduction of 30b using,for example, such reducing agents as lithium aluminum hydride, ordiborane, preferably in an appropriate solvent such as ether , e.g.,THF, dioxane, or diethyl ether. ##STR12##

Even more complex dihydrobenzofuran-based compounds can be obtained byfurther modification of the analogs described above. Any of thecompounds containing the methyl ether groups at the 4-position of thedihydrobenzofuran skeleton can be cleaved by one of the boron orthiolate reagents mentioned supra. Sometimes, as in the limonene series,it may be desirable to carry out the demethylation of the methyl etherwith the carboxylic acid group at the 7-position protected as its alkylester (See, e.g., Scheme 1). Open positions in the phenyl ring may bemetalated and functionalized as disclosed previously. The double bond ofcompound 20a can be hydroxylated by transition metal oxide reagents suchas potassium permanganate or osmium tetroxide. The combination ofmethylether cleaveage and hydroxylation of 20a, for example, would givecompound 24a. Alternatively, compound 24a may be obtained bydemethylation of intermediate 27c to the free phenol. In thoseinstances, as in here, where an alkyl ##STR13## ester is employed toprotect the carboxylic acid group at the 7-position, an alkaline,hydrolysis step may be necessary to obtain the free carboxylic acid(Scheme 1, step i). Other modifications can be envisioned which do notdepart significantly from the scope and essence of the products of thepresent invention. Transformations such as epoxidation of the doublebond, hydroxylation at the allylic position of the double bond, oxidatecleavage of the double bond, hydroformylations, and modifications of theproducts obtained therefrom are but a few non-limiting examples.

5.2.3. PREPARATION OF COMPOUNDS OF GENERAL FORMULA6-CARBOXYL-4-SUBSTITUTED SPIRO[BENZOFURAN -2 (3H) -CYCLOHEXANES ]

6-carboxyl spiro[benzofuran-2(3tt)-cyclohexane] molecules that are alsosubstituted at the 4 position to form a series of ether substitutedderivatives can be prepared as shown in Schemes 3 and 4 below and asmore fully described in Section 7 infra. ##STR14## These compounds andthe salts thereof exhibit complement inhibition as manifested byinhibition of complement mediated hemolysis. Other complement-inhibitoryproperties of these compounds can be evaluated by known techniques asdescribed in Sections 6.6.1 and 10.2 to 10.4 and by numerous complementassay techniques that are known in the art. Substituents for R inposition 4 of compounds of the general formula 4 include a hydrogen atomor a lower alkyl group (e.g. CH₃ --, CH₃ CH₂ --, n-Bu-), afunctionalized lower alkyl group (e.g. HOCH₂ CH₂ --), a benzyl orsubstituted benzyl group, a phenyl or substituted phenyl group (e.g. C₆H₅ CH₂ --, C₆ H₅ --, p-NO₂ C₆ H₄ --, p-CHOC₆ H₄ --, p-NO₂ C₆ H₄ --, andp-NH₂ C₆ H₄ --). In addition the OR at position 4 of compounds of thegeneral formula 4 can instead be H--. Preferred substitutions atposition 4 are exemplified by compounds 44b, 55c and 55e, which are moreeffective in inhibiting complement mediated hemolysis than is K76COOH.

5.2.4. PREPARATION OF COMPOUNDS OF GENERAL FORMULA 6, 7 -DISUBSTITUTED-4-METHOXYSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANES]

6,7-disubstituted spiro[benzofuran-2(3H)-cyclohexane] molecules can beprepared as shown in Schemes 5 and 6 below and as more fully describedin Section 8 infra. ##STR15##

The naturally occurring compound K76 has disubstituted formyl groups inpositions 6 and 7 of the ring D as shown below. Its oxidized derivativeK76COOH has a carboxyl group at position 6 and a formyl group atposition 7. ##STR16##

The compounds of this section differ from K76, since they representdisubstituted BCD analogues lacking the A ring structure of K76. Inaddition the compounds of this section can also be substituted atposition 4 of the D ring to form trisubstituted BCD analogues. These di-and tri-substituted compounds and the salts thereof exhibit complementinhibition as manifested by inhibition of complement mediated hemolysis.Other complement-inhibitory properties of these compounds can beevaluated by known techniques as described in Sections 6.6.1 and 10.2 to10.4 and by numerous complement assay techniques that are known in theart.

The R₁ and R₂ groups can be any combination of the following: a hydrogenatom, a carboxylic acid group, a formyl group; a hydroxymethyl group, anN-(lower alkyl) carbamoyl group, a trifluoroacetyl group, a carbalkoxygroup, a halide group, a vinyl group, a substituted vinyl group havingup to 10 carbon atoms, an alkylidene group having up to 20 carbons, analiphatic acyl group, a substituted aliphatic acyl group, an aromaticgroup, a substituted aromatic acyl group, a trifluoroacetyl group, asulfamoyl group, an N-acylcarbamoyl group, a tetrazole group, a tertiaryaliphatic amine group, an oxazoline group, an amidine group, or ahydrazone group.

Specific substitutions for R₁ and R₂ groups include --CHO, --CH₂ OH,--COOH, COCF₃, SO₂ NH₂ and tetrazole, oxazoline, imide or CH₂ NMe₂derivatives. Substitutions at positions 6 and 7 can be cyclic compoundsas exemplified by compounds 62 and 68. The presence of polar groups inthe 6 and 7 positions appears to affect complement inhibition activity,perhaps because such polar groups interact with regions of thecomplement receptors.

Specific compounds include compounds of general formula 4 in which R canvary generally as described above; R₁ is a carboxyl group or abioisosteric acid group (such as sulfonamide, imide, or tetrazole) or abioisosteric basic group (such as a tertiary aliphatic amine, oxazoline,amidine, or hydrazone), or a bioisosteric neutral group (such astrifluoroacetyl); R₂ is a formyl group or a bioisosteric group such asmethyl ketone (acetyl), other alkyl ketone, aryl ketone or other similargroup.

A preferred organic compound of the compositions of this invention isthe 4,6,7 trisubstituted spiro[benzofuran-2(3)H cyclohexane] such ascompound 68(6-carboxy-7-formyl-4-methoxyspiro[benzofuran-2(3H)-cyclohexane]) and6-carboxy-7-formyl-4-phenoxy-spiro[benzofuran-2(3H)-cyclohexane]. In aspecific embodiment, compound 68 exhibits a relatively large amount ofcomplement inhibition in the hemolysis assay. Even more inhibitorycompounds can be produced by combining the optimal subsitutions at the 4position with the 6,7 disubstitutions present in 68. In a preferredembodiment, a phenoxy group is substituted at the 4 position.

5.3. DEMONSTRATION OF COMPLEMENT INHIBITION

The compounds of the invention can be assayed by any techniques known inthe art in order to demonstrate their complement inhibiting activity.Such assays include but are not limited to the following in vitro testsfor the ability to inhibit complement system activity or to selectivelyinhibit the generation of complement-derived peptides (See Sections 6.6and 10.3 for specific examples):

(i) measurement of inhibition of complement-mediated lysis of red bloodcells (hemolysis);

(ii) measurement of ability to inhibit formation of C5a and C5a des Argand/or measurement of ability to inhibit formation of C3a and C3a desArg;

(iii) inhibition of alternative pathway mediated hemolysis.

Those compounds which are demonstrated to have significantcomplement-inhibiting activity can be therapeutically valuable for thetreatment or prevention of diseases or such as those described inSection 5.5, infra.

5.4. IMMUNOSUPPRESSIVE ACTIVITY

The compounds of the present invention can inhibit immune activity. Inparticular, the compounds of the invention inhibit cell-mediated immunefunction. For example, the compounds can suppress natural killeractivity, inhibit the proliferation of peripheral blood lymphocytes,and/or inhibit the activation of T lymphocytes in PBL culture.

Any procedure known in the art may be employed to demonstrateimmunosuppressive activity. Such procedures include but are not limitedto in vitro assays for inhibition of natural killer lysis of targetcells, inhibition of proliferation of peripheral blood lymphocytes orinhibition of cell surface interleukin-2 receptor expression. Specificembodiments of assay procedures which can be used are detailed in theexamples sections infra (See Subsections 6.7.1 through 6.7.4).

5.5. THERAPEUTIC USES OF THE COMPOUNDS OF THE INVENTION

The compounds of the invention which exhibit complement and/or immuneactivity inhibition have therapeutic value in the prevention ortreatment of various immune or inflammatory diseases or disorders. Thecompounds of the invention may be administered to a patient fortreatment of an immune disorder involving undesirable or inappropriatecomplement activity. In particular, an effective dose of an inhibitivecompound of the invention may be therapeutically applied to ameliorateor to prevent a detrimental effect caused by the activity of a componentof the complement system (e.g., C5a) or an inappropriately reactiveimmune system. An effective dose of the compound of the invention forthe treatment of a disorder involving undesirable or inappropriatecomplement activity or an immune disorder can be determined by standardmeans known in the art taking into account routine safety studies,toxicity studies, dose concentration studies and method of delivery,e.g., bolus, continuous or repeated. In a particular embodiment, a doseof about 0.01 to about 500 mg/kg can be administered.

The diseases or disorders which may be treated by the compounds of theinvention include but are not limited to those listed in Table III,supra. In particular, those disorders associated with extended zones oftissue destruction due to burn--or myocardial infarct--induced trauma,and adult respiratory distress syndrome (ARDS), also known as shocklung, can be treated by administration of an effective amount of thecompounds.

Detrimental nonspecific activation of the complement system, orunfavorable activation by the alternative pathway, can also be preventedor treated by compounds of the invention. In specific embodiments, suchcompounds can ameliorate the acute pathological changes induced byspecific or non-specific proteolytic processing of C5.

The compounds of the invention may also be used to modulate biologic orimmune functions directly or indirectly mediated by the complementsystem, which can include but are not limited to those functions listedin Tables I and II, supra, and the in vivo correlates of the in vitrofunctions therein.

In particular embodiments, the inhibitive compounds can be used to treatinflammation associated with, for example, kidney stones, systemic lupuserythematosis (SLE), nephrotoxic glomeronephritis, or multiple sclerosis(See, e.g., Experimental Allergic Encephalomyelitis. A Useful Model forMultiple Sclerosis, A Satellite Conference of the International Societyof Neurochemists, Jul. 16-19, 1983, University of Washington, Seattle,Wash.; Miyazaki, W. et al. Microbiol. Immunol. 1980, 24, 1091; Konno, S.and Tsurutuji, S. Sr. J. Pharmacol. 1983, 80, 269).

In yet another embodiment, the compounds of the invention can beadministered for treatment of tissue damage due to myocardial ischemiaand reperfusion, resulting from neutrophils attracted by and activatedby the complement system.

The compounds of the invention may also be administered for theprevention or treatment of diseases or disorders caused or accompaniedby increased lymphocyte or natural killer activity, including but notlimited to atrophic gastritus, thyroiditis, allergic encephalomyelitits,gastric mucosa, thyrotoxicosis, autoimmune hemolytic anemia, pemphigusvulgaris, sympathetic opthalmia, delayed-type hypersensitivity,rejection of allografts, graft-host reaction, organ transplantrejection, other autoimmune disorders, and drug allergies. They can alsobe used to alleviate the adverse effects of complement activation causedby therapeutic intervention. such as tissue plasminogen activatortherapy or cardiopulmonary bypass.

Pharmaceutical compositions comprising the inhibitive compounds or thesalts thereof are provided by the present invention. Such compositionscomprise a therapeutically effective amount of the compound and apharmaceutically acceptable carrier. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, and water. Apharmaceutical kit comprising one or more containers filled with one ormore of the ingredients of the pharmaceutical composition is also withinthe scope of the invention.

Various delivery systems (e,g-, encapsulation in liposomes,microparticles, or microcapsules, conjugation to specific molecules) areknown and can be used for therapeutic delivery of the compounds. Methodsof administration include but are not limited to oral, intradermal,transdermal, intravenous, subcutaneous, intramuscular, intraperitoneal,and intranasal routes. Such administration can be done in either bolusor repeat doses or continuously by infusion for instance.

A further embodiment of this invention includes the combined therapythat can be obtained by treating patients with disorders (e.g.myocardial infarction patients) that are routinely treated withthrombolytic agents such as tissue plasminogen activator, streptokinaseor urokinase with a combination of the compounds of this invention andthe routinely administered thrombolytic compounds or a fibrinolyticallyactive fragment, derivative, or modified version thereof. The usefulnessof such a combined therapy derives from the observation that thecomplement system is activated in disorders such as myocardialinfarction or bypass surgery. The efficacy of a combined treatment couldbe substantially better than the thrombolytic treatment alone due to theability of the complement inhibitory compounds to modulate theinappropriate and damaging complement activation. The administration ofthe thrombolytic and complement inhibitory compounds can be simultaneousor sequential or in different dose forms including combinations of oraldose forms with injectables to name just a few.

The invention can be better understood by referring to the followingexamples which are given for illustrative purposes only and are notmeant to limit the invention.

6. EXAMPLES 6.1. 2- (1'-CYCLOHEXENYL) METHYL-3-METHOXYMETHOXYANISOLE(9a)

The 3-methoxymethoxyanisole 7, was obtained in 96% yield by thefollowing procedure. A mixture of finely powdered anhdryous potassiumcarbonate (K₂ CO₃ ; 2.0 equiv) and 3-methoxyphenol (1.0 equiv) in dryacetonitrile was stirred at 0° C. for 15 minutes under nitrogen. Toensure that the reaction pH remained above six, 100 ml of acetonitrilewere used per gram of phenol substrate. A catalytic amount of 18-crown-6(0.12 equiv) was added, and the mixture was stirred an additional 15minutes at 0° C. Neat chloromethyl methyl ether (1.5 equiv) was thenintroduced slowly. The suspension was allowed to warm up to ambienttemperature and stirred for 6 hours. After this time, the mixture wasrecooled to 0° C., and one-half the original quantities of K₂ CO₃,18-crown-6, and CH₃ OCH₂ Cl were added. After another 4 hours ofstirring at room temperature, the suspension was filtered and thefiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl either (Et₂ O), washed with 5% sodium hydroxide (3×50ml), concentrated in vacuo, and distilled under reduced pressure.Compound 7 was obtained as a clear colorless liquid. Bp 45° C. (0.15 mmHg). ¹ H NMR (90 MHz, deuteriochloroform) δ 7.16 (1H, m), 6.61 (3H, m),5.16 (2H, s), 3.79 (3H, s), and 3.49 (3H, s) in ppm downfield from TMS.¹³ C NMR (CDCl₃) δ 160.5,m 158.2, 129.7, 108.2, 107.3, 102.5, 94.3,55.9, and 55.1 in ppm downfield from TMS.

n-BuLi (1.1 equiv) was added slowly to a THF solution of compound 7 (1.0equiv) and TMEDA (1.1 equiv), at 0° C. under a nitrogen atmosphere. Thesolution was stirred at room temperature for 2-5 hours and then cooledto -78° C. Cuprous iodide (1.2 equiv.) was added all at once. The lightgray suspension was warmed up to -40° C. and after stirring for 1.5hours, turned into a green-gray color. The copper reagent was cooled to-78° C. and allowed to react with a THF solution of freshly-preparedallylic bromide 6a (1.3 equiv). The reaction mixture was allowed to warmup to ambient temperature gradually and stirred for up to 72 hours. Themixture was quenched and washed with a saturated aqueous solution ofsodium bicarbonate until the aqueous layer became colorless. The organiclayer was dried by passage through a plug of potassium carbonate andconcentrated in vacuo to give a dark orange oil. The crude product wasdistilled under reduced pressure to provide compound 9a in 69% yield. Bp105° C. (0.15 mm Hg). Anal. Caqlcd. for C₁₆ H₂₂ O₃ : C, 73.25; H, 8.45.Found: C, 73.13; H, 8.50. ¹ H NMR (90 MHz, CDCL₃) δ 7.03 (1H, t, J=8Hz),6.71 (1H, d, J=8 Hz), 6.54 (1H, d, J=8 Hz), 5.23 (1H, broad s), 5.13(2H, s), 3.77 (3H, s), 3.43 (3H, s) 3,32 (3H, broad s) , 1.95 (4H, m) ,and 1.57 (4H, m) in ppm downfield from TMS. ¹³ C NMR (75 MHz, CDCl₃) δ158.5, 155.8, 136.3, 126.7, 120.0, 118.1, 107.0, 104.6, 94.3, 55.8(2C's), 30.9, 28.8, 25.3, 23.1, and 22.6 ppm downfield from TMS. IR(neat) 2940, 2840, 1595, 1470, 1440, 1260, 1160, 1105, 1070, and 1025cm⁻¹.

6.2. 3- (1'-CYCLOHEXENYL) METHYL-2-HYDROXY-4-METHOXYBENZOIC ACID (12a)

Compound 9a was metalated at the 4-position by the following procedure.A hexane solution of 9a (1.0 equiv) and TMEDA (1.1 equiv) was treatedwith a hexane solution of n-BuLi (1.1 equiv) added gradually at 0° C.under an atmosphere of nitrogen. The solution was stirred at roomtemperature for 3 hours and then cooled to -78° C. ##STR17##

The aryllithium reagent Li-9a prepared by the above route was thenexposed to a stream of dried carbon dioxide gas bubbled through the -78°C. solution for 0.5 hour. Carrying out this reaction at 0° C. cuts theresulting yield in half. The mixture was allowed .to warm up to roomtemperature while maintaining a steady stream of gas. The mixture waspoured into water and extracted a few times with 5% aqueous sodiumhydroxide, then acidified to pH 1 with concentrated hydrochloric acid,and then extracted into ether..The crude product was back-extracted intoEt₂ O and the combined organic layers dried over magnesium sulfate. Thesolvent was evaporated and the residue redissolved in a minimum ofboiling ether. The warm solution was allowed to cool slightly, and thenhexane was added to the point of cloudiness. The mixture was thenallowed to stand in the freezer. An off-white solid with a melting pointof 161°-163° C. was harvested (66% yield). Anal. Calcd. for C₁₅ H₁₈ O₄ :C, 68.69; H, 6.92. Found: C, 68.75; H, 6.95. ¹ H NMR (90 MHz,acetone-d.sub. 6) δ 7.81 (1H, d, J=9 Hz) , 6.62 (1H, d, J=9 Hz) , 5.26(1H, broad s) , 4.26 (2H, broad s), 3.90 (3H, s), 3.29 (2H, broad s),2.01 (4H, m) , and 1.57 (4H, m) in ppm downfield from TMS. ¹³ C NMR(acetone-d-₆) δ 173.0, 164.3, 162.1 (2C's), 136.5, 130.6, 121.0, 115.9,106.5 (2C's), 103.3, 56.3, 30.0, 29.4, 25.8, 23.8, 23.2 (2C's). IR (KBr)1650, 1610, 1500, 1455, 1265, 1185, and 1090 cm⁻¹.

6.3. 3-(1'-CYCLOHEXENYL)METHYL-2-HYDROXY-4-METHOXYBENZALDEHYDE (12b)

A solution of aryllithium reagent Li-9a, prepared in situ by theprocedure described in Section 6.2, was cooled to -12° C. and treatedwith neat N,N'-dimethylformamide (1.5 equiv) added all at once. Themixture was stirred at room temperature for 3 hours. The mixture wasthen poured into water, saturated with sodium chloride, and extractedwith Et₂ O. The organic layers were combined, dried (MgSO₄), andconcentrated in vacuo; the residue was recystallized from ether-hexane(See Section 6.2). The product was purified by column chromatography(silica, ether/hexane eluent). Mp 48°-49° C. Anal. Calcd. for C₁₅ H₁₈ O₃: C, 73.15; H, 7.37. Found: C, 73.22; H, 7.40. ¹ H NMR (CDCl₃) δ 11.42(1H, broad s), 9.64 (1H, s), 7.33 (1H, d), 6.51 (1H, d), 5.23 (1H, broads), 3.83 (3H, s), 3.26 (2H, broad s), 1.96 (4H, m), and 1.56 (4H, m) inppm downfield from TMS. ¹³ C NMR (CDCl₃) δ 194.7, 164.6, 161.3, 135.5,133.8, 120.6, 115.9, 15.6, 103.1, 55.9, 29.9, 28.8, 25.2, 23.0, and 22.4in ppm downfield from TMS. IR (KBr) 2930, 2840, 1625, 1495, 1255, 1100,800, and 640 cm⁻¹.

6.4. 7-CARBOXY-4-METHOXYSPIRO[BENZOFURAN-2(3H) -CYCLOHEXANE](11a)

A benzene solution of compound 12a (Section 6.2) was treated with dryAmberlyst ion-exchange resin (approximately 3-4 g per gram of substrate,dried at 110° C. under high vacuum) added in one portion. The mixturewas stirred for up to 24 hours at room temperature and then filtered.The resin beads were washed thoroughly with fresh benzene and methylenechloride. The flitrates were combined, washed with water, dried overmagnesium sulfate, and concentrated in vacuo. The product 11a waspurified in 85% yield by column chromatography. Mp 192°-194° C. Anal.Calcd. for C₁₅ H₁₈ O₄ : C, 68.69; H, 6.92. Found: C, 68.52; H, 6.99. ¹ HNMR (CDCl₃) δ broad roll centered at about 12.0 (1H), 7.85 (1H, d, J=9Hz), 6.50 (1H, d, J=9 Hz), 3.89 (3H, s), 2.94 (2H, s), and 1.69 (10H,broad m) in ppm downfield from TMS. ¹³ C NMR (CDCl₃) 164.5, 160.6,158.7, 132.7, 113.6, 106.2, 104.5, 94.2, 55.7, 38.0, 37.1 (2C's), 24.9,and 23.2 (2C's) in ppm downfield from TMS. FIG. 1 shows the infrared(KBr) spectrum. IR (KBr) 1660, 1615, 1445, 1435, 1385, and 1100 cm⁻¹.Cyclization was also accomplished by heating the phenol precursor ineither a 50/50 4N hydrochloric acid/isopropanol solvent mixture or aboron trifluoride etherate solution in THF. This latter cyclizationprocedure is not recommended for any phenol precursor other than 12a,however. ##STR18##

6.5. 7-FORMYL-4-METHOXYSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE](11b)

The phenol precursor 12b was treated with Amberlyst resin as describedin the previous section. The product 11b was isolated and purified bycolumn chromatography (83% yield). Mp 62°-63° C. Anal. Calcd. for C₁₅H₁₈ O₃ : C, 73.15; H, 7.37. Found: C, 73.22; H, 7.40. ¹ H NMR (CDCl₃) δ10.16 (1H, s), 7.66 (1H, d, J=9 Hz), 6.46 (1H, d, J=9 Hz), 3.88 (3H, s),2.88 (2H, s), 1.4--2.0 (10H, broad m).

Compound 11b is easily transformed to 11e by a metal hydride reductionstep or other means well-known in the art.

6.6. COMPLEMENT INHIBITION

The dihydrobenzofuran and spirobenzo-furancyclohexane compounds of thegeneral formulae 3, 4, the synthetic intermediates of the generalformula 5, and the salts thereof of the present invention were assayedfor their ability to inhibit complement by the methods described infra.

6.6.1. DEMONSTRATION OF INHIBITION OF C3a AND C5a PRODUCTION

The ability to inhibit complement was tested by assaying for specificinhibition of C3a and C5a production. For all experiments, a singlehuman serum pool, to be used as a source of complement, was aliquotedand stored frozen at -70° C. Human IgG was heat-aggregated, aliquoted,and stored frozen at -70° C. For each experiment, serum aliquots wereequilibrated at 37° C. with varying concentrations of the compoundstested. The classical complement pathway was initiated by the additionof aggregated human IgG. Control samples containing no IgG were alwaysincluded. After a fixed reaction time of 10 minutes (determined in anearlier time-course study to provide a convenient time interval duringwhich the production of C5a or C3a is nearly complete, i.e.,.greaterthan 90%), the levels of the released complement peptides (C5a or C3a)were determined by radioimmunoassay using commercially availableradioimmunoassay (RIA) kits (C5a RIA, UpJohn Cat. No. 3250-02; C3a RIA,UpJohn Cat. No. 3245-01; C5a RIA, Amersham Cat. No. RPA.520; C3a RIA,Amersham RPA.518).

Since a competitive immunoassay was used, complement peptide (C5a andC3a) concentrations varied inversely with the counts. The Counts Bound(CB) for a sample was defined as the total counts (in counts per minute,cpm) measured in the pellet minus the counts measured in a non-specificbinding (NSB) control. The NSB control was a sample containing onlytracer peptide (¹²⁵ I-labelled) and second precipitating antiserum; itcontained no C5a- or C3a-specific antiserum.

The y-axis in FIG. 2 represents the fraction inhibition. The fractioninhibition is equal to the Counts Bound (CB) for a "sample," less the CBin the "sample with no added compound," divided by the CB for the "noIgG control" less the CB in the "sample with no added compound."##EQU1##

When C5a production is inhibited at concentrations of added compound atwhich C3a production is unaffected, the data suggest that complementinhibition is directed toward the C5 activation step. FIG. 2 isrepresentative of the data plots obtained from the assays describedabove. The results are summarized in Table VI and demonstrate that thecomplement inhibition activities exhibited by some of the compoundsappear to be directed toward inhibition of C5 activation, and that theinhibitory activities are comparable to that of K-76 COONa itself.

                  TABLE VI                                                        ______________________________________                                        IN VITRO COMPLEMENT INHIBITION                                                IN HUMAN SERUM                                                                               IC.sub.50 (mM).sup.a                                           Compound         C3a    C5a                                                   ______________________________________                                        (+)-22a           11    6                                                     (+)-20a          >14.sup.b                                                                            8                                                     (-)-20a           11    6                                                     (+)-23a          >14.sup.b                                                                            >14.sup.b                                             12b               18    9                                                     12a               13    8                                                     11a               22    5                                                     K-76 COONa        --.sup.c                                                                            3                                                     ______________________________________                                         .sup.a The concentration of compound required to inhibit C3a or C5a           production 50% relative to control samples which contained no test            compound.                                                                     .sup.b The inhibition observed at this concentration was less than about      50%.                                                                          .sup.c Only marginal inhibition was observed at a concentration of about      8.5 mM.                                                                  

6.6.2. DEMONSTRATION OF INHIBITION OF COMPLEMENT-MEDIATED HEMOLYSIS

The ability to inhibit complement was also tested by assaying forinhibition of complement-mediated red cell lysis (hemolysis). Theinhibition of hemolysis was determined as a function of compoundconcentration. The compounds to be tested were diluted in 0.1M Hepesbuffer (0.15 N NaCl, pH 7.4), and 50 μl were added to each well of aV-bottom microtiter plate. Human serum, used as the complement source,was diluted 1 to 500 in Hepes buffer, and 50 μl were added to each well.Next, commercially available sheep erythrocytes with anti-sheep antibody(Diamedix Cat. No. 789-001) were used as received and added at 100μl/well to initiate the complement pathway leading to hemolysis. Theplate was incubated for 60 minutes at 37° C. and then centrifuged at500×g for 10 minutes. The supernatants were removed and placed in aflat-bottom microtiter plate. The extent of hemolysis was measured as afunction of the sample absorbance at 410 nm. The maximal absorbance(corresponding to maximal hemolysis), A.sub. max, was obtained from theabsorbance value of an erythrocyte sample containing only human serum,A_(S), less the absorbance of a sample containing only the red cells,A_(O). Thus, A_(max) =A_(S) -A_(O). The difference between theabsorbance of an erythrocyte sample containing both human serum andinhibitive compound, and the absorbance of a cell sample containinginhibitive compound only, was defined as A_(sample). The inhibition, IH,was expressed as the fraction (A_(max) -A_(sample))/A_(max), and IH₅₀was defined as the concentration of inhibitive compound required toproduce a value of IH=1/2.

FIG. 3 shows the plot of inhibition, IH, versus the concentration of 11aadded to the cell samples. The concentration of 11a corresponding toIH₅₀ is approximately 0.9 mM. Table VII summarizes the results ofseveral assays using some of the compounds of the present invention andshows their effectiveness in inhibition of hemolysis.

                  TABLE VII                                                       ______________________________________                                        INHIBITION OF                                                                 COMPLEMENT-MEDIATED HEMOLYSIS                                                 Compound        IHO.sub.5O (mM)*                                                                          N=                                                ______________________________________                                        11a             1.33 (± 0.49)                                                                          10                                                12a             2.10 (± 0.71)                                                                          2                                                 14a             3.0                                                           15a             0.82 (± 0.16)                                                                          2                                                 (+)-20a         0.68 (± 0.03)                                                                          2                                                 (-)-20a         0.38 (± 0.11)                                                                          2                                                 (+)-23a         2.1                                                           (-)-23a         2.4                                                           30a             0.53 (± 0.19)                                                                          23                                                31a             1.45 (± 0.21)                                                                          2                                                 K-76 COONa      0.57 (± 0.17)                                                                          9                                                 ______________________________________                                         *The concentration of compound required to produce a value for hemolysis      inhibition (IH, as defined supra) of 1/2.                                

6.7. IMMUNOSUPPRESSIVE ACTIVITIES

The dihydrobenzofuran and spirobenzofurancyclohexane compounds of thegeneral formulae 3, 4, intermediate 5, and the salts thereof of thepresent invention were tested for their ability to inhibit cell-mediatedimmune activity. The specific activities demonstrated by the testedcompounds included the inhibition of natural killer (NK) activity, theinhibition of peripheral blood lymphocyte (PBL) proliferation, and theinhibition of cell surface interleukin-2 receptor expression asdescribed infra. In addition, the ability of compound 11a to inhibit theproliferation of Chinese hamster ovary (CHO) cells was tested; noinhibition of CHO cells was observed, suggesting that the compoundsinhibitory activities were specific to the lymphoid cells of the immunesystem. The experiments were conducted using in vitro assays fordetermining dose-dependent effects of the compounds of the invention onimmune function.

6.7.1. DEMONSTRATION OF INHIBITION OF NATURAL KILLER ACTIVITY

Compounds were tested for their effects on the ability of peripheralblood mononuclear cells to lyse NK-sensitive target cells, K562. NKactivity was determined using ⁵¹ Cr-labeled K562 erythromyeloid leukemiacells as targets, and normal peripheral blood mononuclear cells aseffector cells, in a four hour cytotoxicity assay. Effector cells wereisolated from fresh blood by Ficoll-Hypaque gradient centrifugation, and100 μl of a 5×10⁵ cell/well suspension were added to each well of aV-bottom micro-titer plate. The compound 11a was diluted in RPMI 1640medium containing 10% fetal calf serum and dispensed at 100 μl per well.Target cells (K562) were labeled for 30 minutes with 100 μCi of ⁵¹ Cr,washed thoroughly and dispensed in a 20 μl volume at 10⁴ cells/well.These cell concentrations resulted in an effector-to-target cell ratioof 50:1. The microtiter plate was centrifuged at 50×g for 5 minutes andincubated in a humidified chamber with 5% CO₂ at 37° C. After 4 hours,100 μl were removed from each well and the radioactivity was measuredwith a LKB 1275 gamma counter. The percent specific lysis was calculatedas follows:

% specific lysis=[(EXP-SR)/(TOTAL-B)]×100

where EXP (experimental value) was obtained using effector and targetcells; SR (the spontaneous release) was obtained from target cellsincubated with media alone; TOTAL release was obtained by hypotoniclysis in water; and B represents instrumental background. Means werecalculated from quadruplicate wells and the standard deviations neverexceeded 10%. Viability of the effector cells incubated with the testcompound was determined by trypan blue exclusion.

The results of the inhibition of natural killer activity for7-carboxy-4-methoxyspiro[benzofuran-2(3H)-cyclohexane] (11a) sodium saltare summarized in Table VIII.

                  TABLE VIII                                                      ______________________________________                                        INHIBITION OF NATURAL KILLER ACTIVITY BY 7-                                   CARBOXY-4-METHOXYSPIRO[BENZOFURAN-2(3H)-                                      CYCLOHEXANE] (11a), SODIUM SALT                                               (11a)                           %                                             Concen-  %          Spont. Release                                                                            Viability                                     tration  Specific   Target Cells                                                                              of Effector                                   (mM)     Lysis      Alone (K562)                                                                              Cells.sup.a                                   ______________________________________                                        0        17         6           95                                            0.4      7          5           95                                            0.8      4          5           95                                            1.6      1          5           95                                            3.2      -1         5           76                                            6.4      18         23           5                                            ______________________________________                                         .sup.a Viability was determined by trypan blue exclusion.                

The data listed in Table VIII indicate that compound 11a can, atappropriate concentrations, effectively inhibit natural killer activity.

6.7.2. DEMONSTRATION OF INHIBITION OF PROLIFERATION OF PERIPHERAL BLOODLYMPHOCYTES

The proliferation of human peripheral blood lymphocytes (PBL) inresponse to phytohemagglutinin (PHA, Wellcome) or anti-CD3 monoclonalantibody (OKT-3, Ortho) was assessed by the incorporation of ³H-thymidine. Compound 11a was diluted in culture media to the desiredconcentrations, human PBL were added to a final concentration of 10⁶cells/ml, and then either PHA (Wellcome) or anti-CD3 (OKT3, Ortho)antibody (final concentration, 1 mg/ml) was added to initiateproliferation. The final volume per sample was 100 μl. The cells wereincubated for 72 hours after stimulation, pulsed with 1 μCi ³H-thymidine per sample for 4 hours, harvested, and counted in ascintillation counter. Separate experiments, conducted on samplesexposed only to varying amounts of 11a without the stimulant, showedthat the PBL were viable, as determined visually by trypan blueexclusion, in the concentration that compound 11a used above. Theresults (FIGS. 4, 5) showed that compound 11a inhibited PBLproliferation ina dose-dependent manner. Half-maximal inhibition wasobserved at about 0.5 mM of 11a when cultures were stimulated witheither PHA (FIG. 4) or anti-CD3 antibody (FIG. 5). Because cellviability was unaffected by the presence of 11a (see supra), theinhibition apparently was not due to cytotoxic effects. Additionalcompounds of the invention were tested for their immunosuppressiveactivities, with the results presented in Table IX.

                  TABLE IX                                                        ______________________________________                                        INHIBITION OF PROLIFERATION OF                                                PERIPHERAL BLOOD LYMPHOCYTES                                                            IP.sub.50 (mM).sup.a                                                            PHA-stimulated                                                                              OKT-3-stimulated                                    Compound    PBL           PBL                                                 ______________________________________                                        11a         0.4           0.5                                                 15a         0.7           0.4                                                 (+)-20a     2.1           1.7                                                 (-)-23a     2.8           2.0                                                 K-76 COONa  0.5           0.5                                                 ______________________________________                                         .sup.a The concentration of compound required to inhibit PBL proliferatio     50% relative to control samples which contained no test compound.        

The results (Table IX) revealed that each of the tested compoundsinhibited PBL proliferation.

Release of cell surface interleukin-2 receptor (IL-2R) or CD8 antigenfrom lymphocytes is a correlate of T cell activation (Rubin et al. J.Immunol. 1985, 135, 3172-77; Rubin et al. Fed. proc. 1985 44,946;Fujimoto, J. et al. J. Exp. Med. 1983, 159, 752-66; Tomkinson, B. et al.2d Annual Conference on Clinical Immunol. Washington, D.C., Oct. 30,1987). In some experiments, the level of IL-2R or CD8 protein releasedinto the supernatant of the PBL cultures was assessed by removingaliquots therefrom just prior to pulsing with ³ H-thymidine.Commercially available enzyme immunoassay kits (CELLFREE™ IL-2R, Cat.No. CK1020, T Cell Sciences, Inc., Cambridge, Mass., or CELLFREE™T8/CD8, Cat. No. CK1040, T Cell Sciences) were used to determine thelevels of the two analytes. The results using compound 11a showed that11a was an inhibitor of both IL-2R (FIG. 6) and CD8 (FIG. 7) proteinrelease, in stimulated PBL cultures.

6.7.3. DEMONSTRATION OF INHIBITION OF CELL SURFACE INTERLEUKIN-2RECEPTOR EXPRESSION

The interleukin-2 receptor (IL-2R) is not detectable on the surface ofresting T cells. Upon activation by specific antigens or mitogens, Tcell proliferation is mediated by an autocrine mechanism wherebyactivated cells secrete interleukin-2 and express cell surface IL-2R(Meuer, S. C. et al. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1509;Tsudo, M., et al. J. Exp. Med. 1984, 160, 612-617; Waldmann, T. A., etal. J. Exp. Med. 1984, 160, 1450-1466).

Compounds were tested for their ability to inhibit T cell activation, asindicated by inhibition of cell-surface IL-2R expression. PBL cultures(1.5 ml; 24-well plates) were stimulated with PHA (1 μg/ml) for 72 hoursin the absence or presence of varying amounts of compound 11a.Subsequently, the cells were stained using fluorescein isothiocyanate(FITC)-labeled anti-IL-2R antibody (Act-T-Set IL-2R, Cat. No. AA2009, TCell Sciences, Inc., Cambridge, Mass.) and analyzed by flow cytometry(Ortho System 30) (Table X).

                  TABLE X                                                         ______________________________________                                        INHIBITION OF CELL SURFACE IL-2R EXPRESSION                                   BY 7-CARBOXY-4-METHOXYSPIRO[BENZOFURAN-2(3H)-                                 CYCLOHEXANE] (11a), SODIUM SALT                                               Concentration Number of Cells                                                                            Percentage                                         of 11a (mM)   Positive for IL-2R                                                                         Positive.sup.a                                     ______________________________________                                        0             4845         96.9                                               0.44          913          18.3                                               1.76          225           4.5                                               ______________________________________                                         Based upon a total cell count of 5000.                                   

The results listed in Table X indicate a dramatic reduction in thenumber of PBL expressing the IL-2R in the presence of 11a. We alsoobserved that the amount of IL-2R cell surface expression was reduced inthe presence of 11a. The data thus demonstrate that compound 11asignificantly suppresses IL-2R cell-surface expression, indicating thatthis compound can effectively inhibit T cell activation in PBL cultures.

6.7.4. LACK OF INHIBITION OF CHO CELL PROLIFERATION

The specificity of compound 11a's inhibitory activity upon immune cellswas demonstrated by assaying for 11a's ability to inhibit theproliferation of Chinese hamster ovary (CHO) cells. Non-confluent CHOcells were allowed to proliferate for 4 hours in a 96-well plate at avolume of 50 μl per well in the absence or presence of compound 11a at aconcentration of 1.8 mM. The cells were pulsed with 0.5 μCi of ³H-thymidine per sample for 4 hours, harvested, and the amount ofincorporated ³ H-thymidine was determined by scintillation counting. TheCHO cells allowed to proliferate in the absence of added compoundyielded a value of 57025 (±7530) CPM. The value for the sample grown inthe presence of 11a was 52549 (±6272) CPM. These values revealed nostatistical difference in the proliferation rate of the two samples.Because the CHO cell line is of the non-lymphoid type, this resultsuggests that the compounds of the present invention do not generallyinhibit mammalian cell proliferation, but inhibit activation and/orproliferation of PBL cultures.

7. EXAMPLES OF 6-CARBOXYL-4-SUBSTITUTED-SPIRO[BENZOFURAN-2 (3H)-CYCLOHEXANES ] 7.1.6-CARBOXYL-4-METHOXYSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE]SODIUM SALT (44a)AND 6-CARBOXYLATE-4-ETHOXY-SPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] SODIUMSALT (44b.)

Preparation of 33a-b. Methyl 3,5-dihydroxybenzoate (10 g, 59.5 mmol) indry acetone (200 ml) was added to dry K₂ CO₃ (8.2 g, 59.4 mmol).Dimethysulfate (for 33a; 7.5 g, 59.5 mmol) was added to the mixture,which was refluxed for 24 h. The mixture was filtered. Ether (200 ml)was added to the solution and was extracted with H₂ O (100 ml×2). Theorganic solution was dried over MgSO₄ ; after removal of solvent, acrude product (9.0 g) was purified by chromatography (1:19 EtOAc/CH₂Cl₂) to afford 33a as a white solid, yield 3.9 g (36%). mp 95°-97° C.;TLC R_(f) 0.58 (1:9 EtOAc/CH₂ Cl₂). NMR (CDCl₃) δ 7.26-7.13 (m,2H), 6.63(m, 1H), 5.90 (s,1H,OH), 3.90 (s,3H), 3.80 (s,3H). For 33b: Theprocedure of 33a was modified by using diethyl sulfate as alkylatingagent. 33b: mp 98°-100° C.; yield, 33%; TLC R_(f) 0.51 (1:9 EtOAc/CH₂Cl₂). NMR (CDCl₃) δ 7.16-7.14 (m, 2H), 6.62 (m, 1H), 5.70 (s,1H,OH),4.07-4.00 (q,2H), 3.90 (s,1H), 1.41-1.38 (t,3H).

Preparation of 34a-b. NaH (50% in mineral oil; 2.2 g, 91.7 mmol) waswashed with hexane (40 ml×2) under N₂. Dry DMF (50 ml) was added and themixture was cooled to 0° C. A solution of 33a (7.6 g, 41.7 mmol) in dryDMF (40 ml) was added slowly to the mixture, and then stirred for 1 h at25° C. The mixture was recooled to 0° C. and a solution of MOMCl (3.7 g,45.8 mmol) in dry DMF (20 ml) was added dropwise. The mixture wasstirred at 25° C. for 3 h. Ether (200 ml) was added to the mixture andwashed with H₂ O (100 ml×4). The ether portion was dried (MgSO₄) andconcentrated to give a pale yellow liquid as 34a; yield 8.2 g (87%). TLCR_(f) 0.44 (CH₂ Cl₂). IR (neat) 2950, 1740, 1600, 1460, 1430, 1320,1240, 1150, 1060, 1020, 770 cm⁻¹. NMR (CDCl₃) Δ7.32-7.31 (m, 1H),7.24-7.23 (m,1H), 6.79 (m, 1H), 5.19 (s,2H), 3.90 (s,2H), 3.83 (s,3H),3.48 (s,3H). For 34b: Procedure for 34a was modified. Yield, 95%; TLCR_(f) 0.42 (CH₂ Cl₂). IR (neat) 2980, 1725, 1600, 1450, 1300, 1240,1150, 1060, 1030, 770 cm⁻¹. NMR (CDCl₃) δ 7.30-7.29 (m, 1H), 7.23-7.22(m, 1H), 6.79-6.78 (m, lH), 5.19 (s,2H), 4.09-4.02 (q,2H), 3.90 (s,3H),3.48 (s,3H), 1.44-1.39 (t,3H) .

Preparation of 35a-b. LiAlH₄ (1.7 g, 44.2 mmol) was suspended in dry THF(150 ml) under N₂ and cooled to 0° C. A solution of 34a (10 g, 44.2mmol) in THF (20 ml) was added dropwise. The mixture was stirred for 3 hat 25° C. Excess LiAlH₄ was carefully decomposed by additional ice. Themixture was filtered through silica gel and rinsed with ether (150 ml).The solution was washed with H₂ O (50 ml×2), dried and concentrated togive a colorless liquid as 35a, yield 6.7 g (76%). TLC R_(f) 0.38 (1:4EtOAc/CH₂ Cl₂). IR (neat) 3400, 2920, 1600, 1460, 1290, 1150, 1050, 925,840cm ⁻¹. NMR (CDCl₃)Δ6.67-6.50 (m,3H), 5.16 (s,2H), 4.65 (s,2H), S,3H),3.50 (S,3H), 2.56 (s,H,OH). 35b was prepared by the same procedure thatused for 35a: yield, 90-92%; TLC R_(f) 0.52 (1:4 Et₂ O/CH₂ Cl₂). IR(neat) 3400, 2940, 600, 1460, 1390, 1280, 1210, 1155, 1140, 1030, 925,850 cm⁻¹. NMR (CDCl₃) δ 6.80-6.57 (m,3H), 5.27 (s,2H), 4.70 (s,2H),4.20-3.97 (q,2H), 3.50 (s,3H), 2.00 (s,H,OH), 1.50-1.33 (t, 3H).

Preparation of 36a-b. To a solution of 35a (2.2 g, 1.1 mmol) andimidazole (1.51 g, 22.2 mmol) in CH₂ Cl₂ (50 ml), was added slowlyTBDMSCl (2.0 g, 13.3 mmol) dissolved in CH₂ Cl₂ (10 ml). The mixture wasstirred at 25° C. for 3 h. The mixture was washed with H₂ O (100 ml×2),dried with (MgSO₄) and concentrated to give a colorless liquid, 3.3 g(94%) as 36a. TLC R_(f) 0.8 (4: 6 hexane/CH₂ Cl₂) . IR (neat) 2940,1600, 1460, 1365, 1300, 1250, 1210, 1190, 1150, 1100, 1055, 1025, 925,835, 765 cm⁻¹. NMR (CDCl₃) δ 6.62-6.61 (m,1H), 6.58-6.56 (m,1H),6.49-6.47 (t,1H), 5.15 (s,2H), 4.69 (s,2H), 3.78 (s,3H), 3.47 (s,3H),0.95 (s, 9H) , 0.10 (s, 6H) . For 36b: It was prepared by the procedurethat used for 36a; yield, 90-95%. IR (neat) 2940, 1600, 1460, 1390,1370, 1290, 1250, 1150, 1100, 1030, 940, 840, 775 cm⁻¹. NMR (CDCl₃) δ6.61-6.59 (m, 1H), 6.56-6.54 (m, 1H), 6.48-6.47 (t,1H), 5.15 (s,2H),4.67 (s,2H) , 4.04-3.97 (q,2H), 3.47 (s,3H) , 1.43-1.37 (t,3H) , 0.94(s,9H), 0.10 (s,6H).

Preparation of 38a-b. To a solution of 36a (8.2 g, 26.2 mmol) and TMEDA(4.6 g, 39.4 mmol) in dry THF (200 ml) under N₂ at 0° C. n-BuLi (12 ml28.8 mmol) was added slowly. The solution was stirred 30 min at 0° C.and then 1.5 h at 25° C. The solution was recooled to -78° C., CuI (7.5g, 39.4 mmol) was added in one portion under positive N₂ stream. Themixture was stirred for 1.5 h at -78° C. to -40° C. The mixture wasrecooled to -78° C. bromide 37 (5.5 g, 31.5 mmol) in THF (20 ml ) wasadded dropwise, and the mixture was stirred 4 h from -78° C. to 25° C.Ether (200 ml) was added to the solution and washed with 20% NH₄ OHsolution until the aqueous layer was no longer blue, dried andconcentrated to give a brown liquid (10.4 g). Purification bychromatography (1:19 EtOAc/hexane) afforded a colorless liquid asproduct 38a (8.7 g, 82%). TLC R_(f) 0.70 (1:9 EtOAc/hexane). IR (neat)2930, 1610, 1590, 1460, 1430, 1260, 1200, 1100, 1080, 1030, 840, 780cm⁻¹. NMR (CDCl₃) δ 6.68 (s,1H), 6.61 (s,1H), 5.22-5.18 (m, 1H), 5.13(s,2H), 4.71 (s,2H), 3.79 (s,3H), 3.44 (s,3H), 3.26 (s,2H), 1.97-1.88(m,4H), 1.62-1.46 (m,4H), 0.94 (s,9H), 0.10 (s,6H). For 38b: Preparationwas essentially that used for 38a. Yield, 60-62%. TLC R_(f) 0.66 (1:9EtOAc/hexane). IR (neat) 2940, 1610, 1590, 1440, 1390, 1370, 1260, 1200,1150, 1100, 840, 770 cm⁻¹. NMR (CDCl₃) δ 6.67 (s,1H), 6.58 (s, 1H) ,5.25-5.22 (m, 1H) , 5.13 (s, 2H) , 4.69 (s, 2H) , 4.03-3.96 (q,2H), 3.44(s,3H) , 3.28 (s,2H), 2.00-1.89 (m,4H), 1.76-1.47 (m,4H), 1.39-1.35(t,3H), 0.94 (s,9H), 0.10 (s,6H).

Preparation of 39a-b. To the solution of 38a (7.2 g, 14.7 mmol) in THF(100 ml), (Bu)₄ NF (1.0M in THF, 21 ml, 21 mmol) was added slowly at 25°C. Solution was stirred at 25° C. for 2 h. Ether (100 ml) was added tothe solution, then washed with 5% HCl (20 ml) and H₂ O(100 ml×2). Theorganic layer was dried and concentrated to give a colorless liquid as39a, yield 6.0 g (90%) . TLC R_(f) 0.53 (2:3 EtOAc/hexane). IR (neat)3380, 3920, 1610, 1590, 1450, 1430, 1400, 1200, 1160, 1110, 1080, 1030,960, 925, 830 cm⁻¹. NMR (CDCl₃) δ 6.70 (s,1H), 6.61 (s,1H), 5.21-5.18(m, 1H), 5.14 (s,2H), 4.62 (s,2H), 3.79 (s, 3H), 3.44 (s,3H), 3.26(s,2H), 2.00-1.87 (m,4H), 1.62-1.48 (m,4H). For 39b: Preparation wasessentially that used for 39a. Yield, 95%. TLC R_(f) 0.58 (2:3EtOAc/hexane) . IR (neat) 3400, 2940, 1610, 1590, 1440, 1390, 1200,1150, 1120, 1070, 1030, 925, 820 cm⁻¹. NMR (CDCl₃) δ 6.69 (s,1H), 6.58(s,1H), 5.30-5.25 (m, 1H), 5.14 (s,2H), 4.59 (s,2H), 4.03-3.96 (q,2H),3.44 (s,3H), 3.28 (s,2H), 2.00-1.88 (m,4H), 1.60-1.46 (m,4H), 1.39-1.35(t,3H).

Preparation of 40a-b. To a mixture of PCC (5.5 g, 25.6 mmol) in CH₂ Cl₂(100 ml) at 25° C. was added 39a (4.6 g, 5.7 mmol) in CH₂ Cl₂ (50 ml)slowly. The mixture was stirred at 25° C. for 4 h more. The mixture wasfiltered through silica gel and rinsed with EtOAc (40 ml). The solutionwas washed with H₂ O (50 ml), dried (MgSO₄) and concentrated to give ayellow liquid which then was purified by chromatography (3:7EtOAc/hexane) to afford a pale yellow liquid as 40a, yield 4.2 g (92%) .TLC R_(f) 0.66 (3:7 EtOAc/hexane). IR (neat) 2920, 1690, 1580, 1450,1430, 1380, 1300, 1200, 1150, 1110, 1070, 1020, 920, 840, 740, 720 cm⁻¹.NMR (CDCl₃) δ 9.89 (s,1H), 7.23 (s,1H), 7.10 (s,1 H), 5.22 (s,2H),5.20-5.18 (m, 1H), 3.86 (s,3H), 3.46 (s,3H), 3.33 (s,2H), 1.98-1.88(m,4H), 1.61-1.48 (m,4H). For 40b: Procedure of preparation wasessentially that used for 40a. Yield, 75-81%. TLC R_(f) 0.56 (1:4EtOAC/hexane). IR (neat) 2920, 1700, 1585, 1440, 1380, 1310, 1200, 1160,1110, 1070, 1030, 960, 920, 845, 740, 720 cm⁻¹. NMR (CDCl₃) δ 9.88(s,1H) , 7.22 (s,1H) , 7.08 (s,1H), 5.25-5.22 (m, 1H), 5.21 (s,2H),4.12-4.05 (q,2H), 3.47 (s,3H), 3.36 (s,2H), 2.00-1.89 (m,4H), 1.60-1.38(m,4H), 1.43-1.39 (t,3H).

Preparation of 41a-b. To a solution of 40a (4.2 g, 14.5 mmol) in2-propanol (30 ml) and THF (15 ml) at 0° C. was added slowly aq. 4N HClsolution (40 ml, 160 mmol) in 2-propanol (10 ml). The solution was thenstirred at 25° C. for 16 h or until disappearance of 40a (by TLC). Thesolution was extracted with ether (50 ml×3). The ether solution waswashed with H₂ O (30 ml), then dried and concentrated. The crude productwas purified by chromatography (3:7 EtOAc/hexane) to afford 41a as whitesolid, yield g (84%). mp 153°-155° C. TLC R_(f) 0.60 (3:7 EtOAc/hexane). NMR (CDCl₃) δ 9.90 (s,1H), 7.04 (m,2H), 5.78 (s,1H,OH), 5.68-5.65(m,2H), 3.90 (s,3H), 3.48 (s,2H), 2.09-2.01 (m, 4H) , 1.96-1.90 (m, 4H). For 41b: Procedure of preparation was essentially that used for 41a.Yield, 76%; TLC R_(f) 0.34 (1:4 EtOAc/hexane). NMR (CDCl₃) δ 9.86(s,1H), 7.00 (m,2H), 5.80 (s,1H,OH), 5.70-5.66 (m,2H), 4.13-4.06 (q,2H),3.47 (s,2H), 2.05-2.02 (m,2H), 1.92-1.88 (m, 2H), 1.63-1.54 (m,4 H),1.46-1.41 (t,3H).

Preparation of 42a-b. Amberlyst 15 (10 g) was added in one portion tothe solution of 41a (3.3 g, 13.4 mmol) in CH₂ Cl₂ (100 ml). The mixturewas stirred at 25° C. for 6 h. The mixture was filtered and the solutionwas washed with H₂ O (100 ml), dried and concentrated to give a crudeproduct (3 g) which then was purified by chromatography (1:4EtOAc/hexane) to afford 42a as a white solid (2.5 g, 76%). TLC R_(f)0.57 (1:4 EtOAc/hexane). IR (KBr) 2930, 1690, 1600, 1430, 1390, 1350,1325, 1220, 1120, 1030, 920, 830, 805, 745 cm⁻¹. NMR (CDCl₃) δ 9.85(s,1H), 6.93 (s,1H), 6.89 (s,1H), 3.88 (s,3H), 2.94 (s,2H), 1.86-1.64(m,6H), 1.55-1.45 (m,4H). .sup. 13 C NMR (CDCl₃) δ 191.77, 160.53,156.90, 138.19, 121.25, 105.65, 102.81, 90.36, 55.58, 38.48, 37.23,25.05, 22.95. For 42b: Procedure of preparation was essentially thatused for 49a. mp 105°-106° C. Yield, 82%; TLC R_(f) 0.55 (1:4EtOAc/hexane). NMR (CDCl₃) δ 9.84 (s,1H), 6.91 (s,1H), 6.87 (s,1H),4.15-4.08 (q,2H), 2.95 (s,2H), 1.86-1.68 (m,6H), 1.53-1.45 (m,4H),1.45-1.40 (t,3H).

Preparation of 43a-b. To a mixture of aq. 2N NaOH (30 ml) containingAgio (2.4 g, 10.2 mmol) at 50° C., a solution of 49a (1 g, 4.1 mmol) inEtOH (1 ml) and THF (5 ml) was added slowly. The mixture was stirred for6 h at 50° C. The mixture was filtered and the aq. solution was washedwith ether (50 ml). The aq. solution then was cooled to 0° C. andacidified with conc HCl solution The white precipitate that resulted wasextracted with ether, dried and concentrated to afford a white solid as43a, yield 0.75g (71%). TLC R_(f) 0.62 (1:9:10 MeOH/CH₂ Cl₂ /EtOAc). NMR(CDCl₃) δ 7.16 (s,2H), 3.88 (s,3H), 2.94 (s,2H), 1.87-1.65 (m, 6H) ,1.55-1.47 (m, 4H) . For 43b: Procedure for preparation was essentiallythat used for 43a. mp 190°-192° C. TLC R_(f) 0.62 (1:9:10 MeOH/CH₂ Cl₂/EtOAC); yield,69%. IR (KBr) 3300-2400, 1675, 1595, 1430, 1350, 1325,1265, 1210, 1120, 1100, 1030, 955, 860, 770, 740 cm⁻¹. NMR (CDCl₃) δ7.14 (s,2H), 4.15-4.08 (q,2H), 2.95 (s,2H), 1.86-1.65 (m,6H), 1.60- 1.45(m,4H), 1.45-1.40 (t,3H). ¹³ C NMR (CDCl₃) δ 172.17, 160.08, 155.61,130.19, 120.54, 105.39, 104.77, 90.04, 63.80, 38.49, 37.24, 25.09,22.99, 14.84. Anal. Calcd for Cl₁₆ H₂₀ O₄ : C, 69.54; H, 7.30. Found:C,69.40; H.7.35.

Preparation of 44a-b. Sodium hydride (50% in mineral oil, 0.58 g, 24.2mmol) was washed with hexane (30 ml) under N, then dry ether (50 ml) wasadded. A solution of 43a (3.2 g, 12.2 mmol) in ether (150 ml) was addedto above mixture slowly at 25° C. The mixture was stirred at 25° C. for6 h and a white precipitate resulted. The mixture was extracted with H₂O (50 ml). The aqueous solution was collected and freeze dried to give awhite solid (3.2 g, 92%). NMR (D₂ O) δ 7.10 (s,1H), 6.92 (s,1H), 3.85(s,3H), 2.82 (s,2H), 1.70-1.54 (m,6H), 1.44-1.33 (m,4H).

For 44b: Procedure for preparation was essentially that used for 44a.Yield, 75%. NMR (D₂ O) δ 7.09 (s,1H), 6.90 (s,1H), 4.19-4.12 (q,2H),2.89 (s,2H), 1.77-1.60 (m,6H), 1.50-1.40 (m,4H), 1.40-1.36 (t,3H).

7.2. 6-FORMYL-4-HYDROXYSPIRO[BENZOFURAN-2(3H) -CYCLOHEXANE](52)

Preparation of 45. The procedure for the preparation of 45 wasessentially that used for 34 except that two molar equivalent of NaH andMOMCl were used. Yield was 83%; TLC R_(f) 0.65 (1:1 ether/hexane).

Preparation of 46. The procedure for preparation of 46 was essentiallythat used for 35. Yield was 89%; TLC R_(f) 0.27 (1:1 ether/hexane).

Preparation of 47. The procedure for preparation was essentially thatused for 36. 47: colorless liquid; yield, 83-98%; TLC R_(f) 0.80 (1:1ether/hexane). IR (neat) 2960, 600, 1470, 1400, 1370, 1290, 1260, 1140,1080, 1040, 930, 840, 780 cm⁻¹. NMR (CDCl₃) δ 6.92-6.72 (m,3H), 5.26(s,4H), 4.80 (s, 2H) , 3.57 (s, 6H) , 1.00 (s, 9H) , 0.13 (s, 6H) .

Preparation of 48. Procedure for preparation of 48 was essentially thatused for 38. Crude product was purified by chromatography (1:19EtOAc/hexane). 48: a clear liquid; yield, 63-79%; TLC R_(f) 0.46 (1:19EtOAc/hexane), IR (neat) 2940, 1615, 1590, 1455, 1440, 1400, 1370, 1260,1160, 1100, 1050, 930, 840, 780 cm⁻¹. NMR (CDCl₃) δ 6.76 (s,2H),5.25-5.22 (m,1H), 5.15 (s,4H), 4.70 (s,2H), 3.45 (s, 6H) , 3.30 (s, 2H), 2.10-1.96 (m, 4H) , 1.95-1.88 (m, 4H) , 0.95 (s,9H), 0.11 (s,6H).

Preparation of 49. Procedure for preparation 49 was essentially thatused for 39. Yield, 95-100%; TLC R_(f) 0.45 (3:7 EtOAc/CH₂ Cl₂). IR(neat) 3400, 2930, 1620, 1590, 1440, 1290, 1190, 1155, 1110, 1040, 950,920, 840 cm⁻¹. NMR (CDCl₃) δ 6.78 (s,2H), 5.24-5.20 (m, 1H), 5.17(s,4H), 4.63 (s,2H), 3.45 (s,6H), 3.30 (s,2H), 2.10-1.88 (m,4H), 1.73(s,1H,OH), 1.63-1.48 (m,4H).

Preparation of 50. Procedure for preparation 50 was essentially thatused for 40. Crude product was purified by chromatography (3:7ether/hexane). Yield, 70-76%; TLC R_(f) 0.43 (3:7 ether/hexane). IR(neat) 2930, 1700, 1585, 1435, 1380, 1290, 1155, 1105, 1050, 950, 925 cm⁻¹. NMR (CDCl₃) δ 9.89 (s,1H), 7.29 (s,2H), 5.23-5.18 (m,5H), 3.46(s,6H), 3.37 (s,2H), 2.00-1.95 (m,4H), 1.94-1.87 (m,4H). ¹³ C NMR(CDCl₃) δ 191.65, 156.49, 135.74, 135.54, 126.38, 121.11, 108.79, 94.39,51.15, 31.61, 28.95, 25.30, 23.10, 22.46.

Preparation of 51. Procedure for preparation 51 was essentially thatused for 41. Crude product was purified by chromatography (3:7EtOAc/hexane). It should be mentioned that a large amount of expectedproduct decomposed or was trapped on silica gel to give a low yield(28%). For 51: mp 130°-132° C. TLC R_(f) 0.41 (3:7 EtOAc/hexane). IR(KBr) 3460, 3420, 2940, 1690, 1670, 1595, 1450, 1410, 1340, 1200, 1170,1140, 1080, 1050, 1035, 1010, 840, 790, 740 cm⁻¹. NMR (CDCl₃) δ 9.83(s,1H), 6.96 (s,2H), 5.72-5.68 (m, 1H) , 5.67 (s,2H,OH) , 3.47 (s,2H),2.08-2.01 (m,2H), 2.00-1.92 (m, 2H) , 1.67-1.53 (m, 4H) .

Preparation of 52. Procedure of preparation 52 was essentially that usedfor 42. Crude product was purified by chromatography with 3:7(EtOAc/hexane) as eluent. For 52: TLC R_(f) 0.53(3:7 EtOAc/hexane). IR(KBr) 3260, 2940, 1680, 1590, 1460, 1400, 1310, 1280, 1250, 1210, 1175,1110, 1060, 1000, 930, 920, 840, 800, 740 cm⁻¹. NMR (CDCl₃) δ 9.81 (s,1H), 6.88 (s,1H), 6.86 (s,1H), 2.97 (s,2H), 1.89-1.65 (m, 6H), 1.58-1.45(m, 4H) .

7.3. 4-BENZYLOXY-6-FORMYL-SPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (53a)

Preparation of 53a. Sodium hydride (50% in mineral oil, 62 mg, 2.58mmol) was washed with hexane (20 ml) under N₂. Dry DMF (5 ml) was addedand a solution of 52 (0.30 g, 1.29 mmol) in DMF (10 ml) was added slowlyat 25° C. After stirring for 30 min, a solution of benzyl bromide (0.22g, 1.29 mmol) in DMF (5 ml) was added dropwise. The mixture was warmedto 50° C. and stirred for 2 h more. Ether (50 ml) was added to themixture which was then washed with H₂ O(50 ml×3), dried and concentratedto afford a crude product (0.4 g). Purification by chromatography (1:4Et₂ O/hexane) gave a pale yellow liquid as 53a, yield 0.3 g (71%). TLCR_(f) 0.45 (1:4 Et₂ O/hexane). IR (neat) 2940, 2870, 1695, 1600, 1440,1390, 1355, 1330, 1220, 1210, 1150, 1100, 1045, 920, 830, 805, 750, 700cm⁻¹. NMR (CDCl₃) δ 9.85 (s,1H), 7.46-7.34 (m,5H), 7.01 (s,1H), 6.91(s,1H), 5.14 (s,2H), 2.98 (s,2H), 1.87-1.64 (m,6H), 1.56-1.42 (m,4H).

7.4. 4-N-BUTYLOXY-6-FORMYL-[NBU-]SPIRO [BENZOFURAN-2 (3)]-CYCLOHEXANE](53b)

Preparation of 53b. Sodium hydride (50% in mineral oil, 62 mg, 2.58mmol) was washed with hexane (30 ml) under N₂, then dry DMF (5 ml) wasadded. To the mixture, a solution of 52 (0.3 g, 1.29 mmol) in dry DMF(10 ml) was added dropwise. The mixture was stirred for 30 min. Asolution of n-butyl p-toluenesulfonate (0.29 g, 1.29 mmol) in DMF (5 ml)was added dropwise. The mixture was warmed to 50° C. and stirred for 2h. Ether (50 ml) was added to the mixture which was washed with H₂ O (50ml×2), dried and concentrated to give a crude product. Purification bychromatography (1:4 ether/hexane) afforded a pale yellow liquid as 53b,yield 0.27g (73%). TLC R_(f) 0.58 (ether/hexane). IR (neat) 2940, 2860,1690, 1600, 1430, 1380, 1320, 1210, 1100, 1030, 920, 825, 800, 745cm⁻ 1. NMR (CDCl₃) δ 9.84 (s,2H), 6.92 (s,1H), 6.87 (s,1H), 4.07-4.03(t,2H), 2.94 (s,2H), 1.87-1.64 (m,8H), 1.58-1.43 (m,6H), 1.01-0.96(t,3H).

7.5. 6-FORMYL-4-PHENOXYSPIRO[BENZOFURAN-2 (3H) -CYCLOHEXANE] (53c)

Preparation of 53c. Under N₂ condition, a solution of 52 (0.4 g, 1.72mmol) in CH₂ Cl₂ was added to a mixture of triphenylbismuth diacetate(1.16 g, 2.08 mmol) and copper powder (11 mg, 0.17 mmol) in CH₂ Cl₂ (50ml). The mixture was stirred at 25° C. for 24 h, then filtered throughsilica gel and rinsed with 10% EtOAc in CH₂ Cl₂ solution (20 ml). Thesolution was washed with H₂ O(50 ml), dried and concentrated to give thecrude product (0.65 g), which then was purified by chromatograhy (1:4ether/hexane) to afford a colorless liquid as 53c; yield 0.4 g (75%).TLC R_(f) 0.59 (1:4 ether/hexane). IR (neat) 3060, 2940, 2860, 1695,1580, 1485, 1430, 1310, 1215, 1050, 1020, 850, 800, 755, 690 cm⁻¹. NMR(CDCl₃) δ 7.40-6.89 (m, 7H) , 2.93 (s, 2H), 1.90-1.63 (m, 6H) ,1.55-1.42 (m, 4H) .

7.6. 6-FORMYL-4- (2'-HYDROXYETHYLOXY) SPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE] (53d)

Preparation of 53d. Sodium hydride (50% in mineral oil, 0.10 g, 2.15mmol) was washed with hexane (50 ml) under N₂. Dry DMF (15 ml) was addedand a solution of 52 (0.50 g, 1.29 mmol) in DMF (10 ml) was added slowlyat 25° C. After stirring for 30 min, a solution of2-[(tertbutyldimethylsilyl)oxy]ethyl bromide in DMF (5 ml) was addeddropwise. The mixture was warmed to 50° C. and stirred for 3 h more.Ether (50 ml) was added to the mixture, which was then washed with water(50 ml×3). The ether layer was collected, dried and concentrated to givea crude product (0.9 g). Purification by chromatography (1.5: 8.5ether/hexane) gave a pale yellow solid 0.70g (83%). TLC R_(f) 0.50 (1:4ether/hexane). mp 74°-75° C. IR (KBR) 2940, 2860, 1695, 1600, 1440,1390, 1330, 1220, 1140, 1110, 1095, 960, 830, 780 cm⁻¹. NMR (CDCl₃) δ9.84 (s,1H), 6.93 (s,1H), 6.89 (s,1H), 4.13 (t,2H), 3.98 (t,2H), 2.95(s,2H), 1.87-1.45 (m,10H), 0.91 (s,9H), 0.10(s,6H).

The above product (0.31 g, 0.79 mmol ) was dissolved in THF (5 ml), thentetrabutylammonium fluoride (1 ml, 1.0 mmol, 1.0M in THF solution) wasadded, solution was stirred at 25° C. for 2 h. Ether (30 ml) was added,and the solution was washed with water (50 ml). The ether layer wascollected, dried and concentrated to afford a pale yellow liquid.Purification by chromatography (1:4 hexane/ether) gave a pale yellowliquid as 53d. Yield, 0.17g (77%). TLC R_(f) 0.34 (1:4 hexane/ether). IR(neat) 3450(b) , 2940, 2870, 1690, 1600, 1440, 1390, 1325, 1220,1130-1070(b), 1035, 925, 830, 810, 755cm⁻¹. NMR (CDCl₃) δ 9.84 (s,1H),6.93 (s,1H), 6.91 (s,1H), 4.18 (t,2H), 4.00 (t,2H), 2.97 (s,2H),1.87-1.64 (m,6H), 1.49 (s,b,4H).

7.7. 6-FORMYL-4-P-NITROPHENOXYSPIRO [BENZOFURAN-2(3)-CYCLOHEXANE] (53e)

Preparation of 53e. Sodium hydride (50% in mineral oil, 0.10 g, 4.58mmol) was washed with hexane (20 ml) under nitrogen, and dry DMF (50 ml)was added. To the mixture, a solution of 52 (0.50 g, 2.15 mmol) in DMF(15 ml) was addded slowly at 25° C. The mixture was stirred for 30 min.A solution of 1-fluoro-4-nitrobenzene in DMF (10 ml) was added slowly.The mixture was then warmed to 35° C. and stirred for 3 h. Ether (100ml) was added to the mixture after cooling to 25° C. which was washedwith water (50 ml×3). Ether layer was collected, dried and concentratedto give a crude product (0.79 g). Purification by chromatography (1:9EtOAc/hexane) afforded a yellow sticky oil as expected product 53e.Yield, 0.41g (54%). TLC R_(f) 0.32 (1:4 EtOAc/hexane). IR (neat) 2940,2860, 1695, 1570, 1515, 1490, 1430, 1345, 1310, 1240, 1165, 1110, 1065,1050, 1025, 920, 865, 850, 800, 750, 745 cm⁻¹. NMR (CDCl₃) δ 9.86(s,1H),8.26-8.23 (d,1H), 7.06 (s,1H), 7.06-7.03 (m,3H), 2.86 (s,2H), 1.90-1.40(m,10H).

7.8. 6-FORMYL-4-P-FORMYLPHENOXYSPIRO [BENZOFURAN-2 (3H)-CYCLOHEXAN](53f)

Preparation of 53f. Sodium hydride (50% in mineral oil, 103 rag, 4.31mmol) was washed with hexane (20 ml) under N₂. Dry DMF (30 ml) was addedand a solution of 52 (0.40 g, 1.72 mmol) in DMF (5 ml) was added slowlyat 25° C., and a solution of p-fluorobenzaldehyde in DMF (5 ml) wasadded. The mixture was warmed to 50° C. and stirred for 24 h. Aftercooling to 25° C., ether (100 ml) was added, the solution was washedwith water (30 ml×3). The ether layer was collected, dried andconcentrated to give a crude product. Purification by chromatography(1:9 EtOAc/hexahe) gave a pale yellow solid as expected product 53f.Yield, 0.31 g (53%). 53f: mp 127°-129° C. TLC R_(f) 0.53 (1:4EtOAc/hexane). IR (KBR) 2940, 2840, 1680, 1560, 1500, 1440, 1390, 1320,1220, 1170, 1050, 840, 800cm⁻¹. NMR (CDCl₃) δ 9.95 (s,1H), 9.85 (s,1H),7.90-7.87 (d,2H), 7.12 (s,lH) , 7.10-7.07 (d,2 H), 7.04 (s,1H) , 2.87(s,2H), 1.90-1.40 (m, 10H) .

7.9. 6-FORMYLSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE] (53g)

Preparation of 53g. Pyridine (0.34 g, 4.31 mmol) and 52 (0.50 g, 2.15mmol) were dissolved in methylene chloride (50 ml) and cooled at 0° C.under N₂. Triflic (trifluoromethanesulfonic) anhydride (0.40 ml, 2.37mmol) was introduced dropwise by syringe. The solution was stirred for 1h at 0° C. The solution was then washed with 5% aq. HCl (10 ml) andwater (30 ml×3), dried and concentrated to give a crude product, whichwas purified by chromatography (1:9 EtOAc/hexane) to afford the expectedproduct phenol triflate 52. Yield, 0.70 g (90%). Phenol triflate 52: TLCR_(f) 0.74 (1:4 EtOAc/hexane). IR (neat) 2940, 2860, 1710, 1625, 1590,1430, 1300, 1250-1200(b), 1140, 1010, 980, 920, 850, 830, 800, 770 cm⁻¹.NMR ((CDCl₃) δ 9.89 (s, 1H) , 7.25 (s, 2H) , 3.12 (s, 2H) , 1.92-1.44(m,-10n).

Phenol triflate 52 (1.00 g, 2.74 mmol), triphenylphosphine (58 mg, 0.22mmol), palladium acetate (25 mg, 0.11 mmol) and triethylamine (1.66 g,16.4 mmol) were dissolved in DMF (10 ml) and 96% formic acid (0.50 g,10.8 mmol) in DMF (2 ml) was added. The mixture was stirred at 60° C.for 2 h under N₂. The mixture was diluted with water (50 ml) andextracted with ether (50 ml×3). The ether layer was collected, dried andconcentrated to give a brown residue. The crude product was purified bychromatagraphy (1:9 EtOAc/hexane) to give the expected product 53g as apale yellow oil. Yield, 0.29 g (49%). g: TLC R_(f) 0.69 (1:9EtOAc/hexane). IR (neat) 2940, 2860, 1690, 1585, 1490, 1440, 1340, 1310,1270, 1250, 1150, 1030, 950, 920, 870, 800, 785 cm⁻¹. NMR (CDCl₃) δ 9.90(s,1H), 7.36-7.33 (d,1H), 7.28-7.26 (d, 1H), 7.22 (s,1H), 3.02 (s,1H),1.89-1.43(m, 10H).

7.10. 4-BENZYLOXY-6-CARBOXYLSPIRO [BENZOFURAN-2 (3H)-CYCLOHEXANE] (54a);4 -N-BUTYLO XY-6 -CARBO XYLSP IRO [BENZOFURAN-2 (3H)-CYCLOHEXANE] (54b);6-CARBOXYL-4 -PHENOXY SPIRO [BENZOFURAN-2 (3H)-CYCLO HEXANE] (54c);6-CARBOXYL-4- (2'-HYDROXYETHYLOXYSPIRO [BENZOFURAN-2 (3H)-CYCLOHEXANE](54d); AND 6-CARBOXYL-4-P-NITROPHENOXYSPIRO [BENZOFURAN-2(3H]-CYCLOHEXANE] (54e)

Preparation of 54a-e. Procedure for preparation of these compounds wasessentially that used for 43. For 54a: mp 173°-175° C.; yield, 74-83%;TLC R_(f) 0.56 (1:4 hexane/ether). IR (KBr) 3100-2500, 1680, 1600, 1420,1320, 1215, 1095, 950, 735cm⁻¹. NMR (CDCl₃) δ 7.46-7.34 (m,5H), 7.26(s,1H), 7.18 (s,1H), 5.13 (s,2H), 2.97 (s,2H), 1.86-1.65 (m,6H), 1.48(s,br,4H). Anal. Calcd for C₂₁ H₂₂ O₄ : C, 74.53; H, 6.55. Found: C,74.44; H, 6.57.

For 54b: mp 145°-147° C.; yield, 81%; TLC R_(f) 0.62 (1:4 hexane/ether).IR (KBr) 3100-2500, 1670, 1600, 1425, 1320, 1275, 1210, 1100, 1030, 940,865, 765, 745cm⁻¹. NMR (CDCl₃) δ 7.14 (s,2H), 4.06-4.02 (t,2H), 2.94(s,2H), 1.86-1.65 (m,8H), 1.56-1.44 (m,6H), 1.01-0.97 (t,3H). Anal.Calcd for C₁₈ H₂₄ O₄ : C, 71.01; H, 7.95. Found: C, 71.01; H, 7.97 .

For 54c: mp 173°-175° C.; yield, 71%; TLC R_(f) 0.69 (1:4 hexane/ether).IR (KBr) 3100-2500, 1680, 1580, 1480, 1420, 1310, 1205, 1050, 950, 865,765, 685cm⁻¹. NMR (CDCl₃) δ 7.37-6.97 (m,7h), 2.90 (s,2H), 1.90-1.60(m,6H), 1.50-1.40 (m,4H). ¹³ C NMR (CDCl₃) δ 171.45, 160.88, 156.32,153.39, 130.62, 129.86, 123.74, 123.56, 118.44, 112.37, 106.59, 90.34,38.70, 37.11, 25.02, 22.90. Anal. Calcd for C₂₀ H₂₀ O₄ : C,74.05; H,6.22. Found: C, 73.97; H, 6.25.

For 54d: Reaction was stirred for 8 h at 50° C. 54d: mp 163°-164° C.;TLC R_(f) 0.72 (1:19 MeOH/ether); yield, 94%. IR (KBr) 3600-3300(b),2940, 2860, 1690, 1600, 1430, 1325, 1320, 1115, 1070, 960, 755, 725cm⁻¹. NMR (CDCl₃) δ 57.15 (s,1H), 7.14 (s,1H), 4.18 (t,2H), 3.99 (t,2H),2.96 (s,2H) 1.87-1.65 (m,6H), 1.49 (s,b, 4H). ¹³ C NMR (CDCl₃) δ 171.24,160.26, 155.23, 130.28, 120.61, 105.44, 105.36, 90.21, 69.46, 61.43,38.48, 37.23, 25.05, 22.96. Anal. Calcd for C₁₆ H₂₀ O₅ : C,65.74;H,6.90. Found: C,65.58; H,6.96.

For 54e: Mixture was stirred for 24 h at 50° C. Yield, 56%. 54e: mp190°-191° C.; TLC R_(f) 0.80 (ether). IR (KBr) 3100-2500(b) , 1690,1585, 1525, 1490, 1425, 1390, 1310, 1230, 1170, 1115, 1070, 1025, 960,860, 850, 770, 755 cm⁻¹ NMR ((CDCl₃) δ 8.25-8.22 (d,2H), 7.36 (s,lH) ,7.29 (s,1H) , 7.04-7.01 (d,2H), 2.84 (s,2H), 1.90-1.40 (m,10H). ¹³ C NMR(CDCl₃) δ 170.45, 161.92, 161.35, 150.96, 143.03, 131.22, 126.12,124.98, 116.93, 113.98, 108.45, 90.76, 38.66, 37.05, 24.91, 22.81. Anal.Calcd. for C₂₀ H₁₉ NO₆ : C, 65.03; H, 5.18; N,3.79. Found: C, 64.96; H,5.23; N, 3.71.

7.11. 6-CARBOXYLSPIRO[BENZOFURAN-2(3H) -CYCLOHEXANE] (54g); AND4-P-AMINOPHENOXY-6-CARBOXYLSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (54h); 6-CARBOXYL-4-P-CARBOXYLPHENOXYSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (54i)

54g: Mixture was stirred at 60° C. for 4 h. Yield, 84%. mp 154°-155° C.TLC R_(f) 0.82 (ether) . IR (KBR) 3200-2500 (b) , 1680(b), 1590, 1440,1390(b), 1090, 1030, 950, 780, 770cm⁻¹. NMR (CDCl₃) δ 7.62-7.60 (d,1H),7.44 (s,1H), 7.21-7.19 (d,1H), 3.01 (s,1H), 1.89-1.43 (m,10H). Anal.Calcd for C₁₄ H₁₆ O₃ : C, 72.39; H,6.94. Found: C, 72.31; H, 6.95.

54h: 54e (30 mg, 0.81 mmol) dissolved in MeOH (10 ml) and 10% Pd-C (20mg) was added. H₂ gas was bubbled through the mixture for 5 h at 25° C.The mixture was filtered and MeOH was removed to give a brown oil. Thecrude product was purified by preparative TLC plate (1:19 MeOH/CH₂ Cl₂)to afford a pale yellow solid as product 54h. Yield, 10 mg (36%). 54h:mp 193°-195° C. TLC R_(f) 0.49 (1:9 MeOH/CH₂ Cl₂). IR (KBr) 3380, 3320,2940. 2860, 1700, 1600, 1510, 1420, 1320, 1290, 1270, 110, 1200, 1050,950, 920, 770cm⁻¹. NMR (CDCl₃) 6 7.15 (s,1H), 7.00 (s,1H), 6.87-6.84(d,2H), 6.87-6.84 (d,2H), 2.94 (s,2H), 1.88-1.43 (m,10H). MS m/z(relative intensity) 339(100) , 322(2) , 296(5) , 282(7) , 258(29),217(61), 196(11), 167(15), 156(7), 130(9), 108(29), 93(95).

54i. Procedure for preparation of these compounds was essentially thatused for 43. Mixture was stirred for 24 h at 60° C. The crude productwas treated with CH₂ N₂ which then was purified by chromatography (1:4EtOAc/hexane) to give the dimethyl ester of 54i. Yield, 0.18g (51%).Dimethyl ester of 54i: IR (neat) 2940, 2860, 1750, 1590, 1500, 1430,1350, 1310, 1280, 1250, 1220, 1160, 1110, 1080, 1070, 1050, 1030, 1000,920, 880, 850, 770, 690 cm⁻. NMR (CDCl₃) δ 8.03-8.00 (d,2H), 7.26(s,1H), 7.21 (s,1H), 6.98-6.95 (d,2H), 3.91 (s,3H), 3.86 (s,3H), 2.82(s,2H), 1.88-1.39 (m,10H).

The dimethyl ester of 54i (0.15 g, 0.38 mmol) was dissolved in THF (10ml), then a solution of 2N NaOH (5 ml) was added. The solution waswarmed to 50° C. and stirred for 3 h After cooling to 25° C. the aq.solution was rinsed with ether (20 ml) and the aq. solution wasacidified with conc. HCl. A white precipitate resulted, which wasextracted with ether (50 ml×2). After the precipitate was dried andconcentrated, a white solid was resulted as product 54i. Yield, 0.13 g(93%). 54i mp 278°-280° C. TLC R_(f) 0.29 (1:9 MeOH/CH₂ Cl₂). IR (KBr)3200-2400(b), 1690, 1605, 1590, 1505, 1425, 1315, 1280, 1220, 1170,1050, 950, 915, 880, 850, 770, 740, 720cm⁻¹. NMR (MEOH-d₄) δ 8.05-8.02(d,2H), 7.18 (s,1H), 7.15 (s,1H), 7.03-7.00 (d,2H), 2.84 (s,2H),1.86-1.43 (m, 10H). ¹³ C NMR (MeOH-d4) δ 169.21, 168.97, 162.48, 162.13,153.28, 133.91, 133.13, 126.71, 124.86, 118.09, 114.23, 108.04, 91.47,39.76, 37.98, 26.09, 23.90. Anal. Calcd for C₂₁ H₂₀ O₆ : C, 68.47; H,5.47. Found: C, 68.22; H, 5.48.

7.12. SODIUM SALTS OF 4-BENZYLOXY-6-CARBOXYLSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (55a); 4-N-BUTYLOXY-6-CARBOXYLSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE] (55b); 6-CARBOXYL-4-PHENOXYSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE] (55c); 6-CARBOXYL-4-(2'-HYDROXYETHYLOXYSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (55d);6-CARBOXYL-4-P-NITROPHENOXYSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (55e);6-CARBOXYLSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE] (55g); AND6-CARBOXYL-4-P-CARBOXYLPHENOXYSPIRO [BENZOFURAN-2(3H) -CYCLOHEXANE](55i)

Preparation of 55a-e,g,i. Procedure for preparation of these compoundswas essentially that used for 44. NMR spectra for 55a-g are listed asbelow. 55a: NMR (D₂ O) δ 7.21-6.88 (s,7H), 4.85 (s,2H), 2.56 (s,2H),1.43-1.06 (m, 10H). 55b: NMR (D₂ O) δ 7.10 (s,1H), 6.90 (s,1H), 4.13(t,2H) , 2.93 (s,2H) , 1.79-1.67 (m,8H) , 1.50-1.41 (m,6H) , 0.93(t,3H). 55c: NMR (D₂ O) δ 7.20-6.80 (m,7H), 2.61 (s,2H), 1.60-1.12(m,10H). 55a: NMR (D₂ O) δ 7.07 (s,1H), 6.92 (s,lH), 4.18 (t,2H), 3.93(t,2H), 2.96 (s,2H), 1.68 (s, b, 6H), 1.44(s, b, 4H). 55e: NMR (D₂ O) δ8.10-8.07 (d,2H), 7.13 (s,1H), 7.11 (s,1H), 6.98-6.95 (d,2H), 2.68(s,2H), 1.70-1.23 (m,10H). 55 i: NMR (D₂ O) 7.90-7.87 (d,2H), 7.14(s,1H), 7.12 (s, 1H), 7.00-6.97 (d,2H), 2.76 (s,2H), 1.70-1.27 (m,10H).55g: NMR (D₂ O) δ 7.45-7.42 (d,1H), 7.23-7.20 (m,2H), 2.93 (s,2H), 1.65(s, b, 6), 1.41 (s,b, 4H) . ¹³ C NMR (D₂ O) δ 177.63 , 160.05 , 139.25,133.68, 127.83, 124.60, 112.09, 93.64, 42.45, 38.96, 27.15, 25.37.

8. EXAMPLES OF6,7-DISUBSTITUTED-4-METHOXYSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANES]

The melting points were measured with a Thomas Hoover apparatus, and areuncorrected; the infrared spectra were obtained with the aid of a PerkinElmer 281B spectrophotometer, the ¹ H NMR spectra were recorded indeuteriochloroform, unless another solvent is specified, either at 90MHz, in a Varian EM 390 apparatus, or at 300 MHz in a Varian VXR 300spectrometer. The ¹³ C NMR were recorded at 75.4 MHz in a Varian VXR 300spectrometer. All the signals are reported in ppm, downfield fromtetramethylsilane, used as internal reference. The multiplicities of thesignals are abbreviated as: s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet, b=broad signal. The low resolution mass spectra wereobtained from a Finnigan 3221-F200 spectrograph, at 70 eV. The elementalanalyses were done by Atlantic Mirolab, Inc. (Norcross, Ga.). The HRMSwas obtained at Massachusetts Institute of Technology.

All the solvents and chemicals used were of good quality. In severalcases, some of them were purified and/or dried following preestablishedprocedures. All the reactions were carried out under a dry nitrogenatmosphere. The column chromatographies were done using MN Silica gel60, under atmospheric pressure, using different solvent systems(hexane-ether, hexane-ethyl acetate), in which increasing quantities ofthe second solvent were periodically added to the first solvent, or on achromatotron. One percent acetic acid was added during thechromatographies of carboxylic acids.

8.1. SYNTHESIS OF 7-CARBOXY-6-FORMYL-4-METHOXYSPIRO[BENXOFURAN-2(DH)-CYCLOHEXANE (62)

Preparation of 58: A solution of n-butyllithium in hexane (32.67 ml,65.34 mmol) was added dropwise to a cold solution (0° C.) of3,5-dimethoxybenzyl alcohol (56, 5 g, 29.7 mmol) and TMEDA (5.67 ml,35.64 mmol) in THF (300 ml). After stirring at 0° C. for 15 min and atroom temperature for 90 min, the reaction was cooled to -45° C. andcopper (I) iodide was added (7054 mg, 37.13 mmol). One hour later, theelectrophile 57 [prepared as described in J. Heterocyclic. Chem. 26,879-891 (1989)]was added via syringe. After stirring overnight at roomtemperature, concentrated ammonium hydroxide (100 ml) was added, and thereaction products were extracted with ethyl acetate (4×150 ml). Thecombined organic phases were dried over MgSO₄, the solvent wasevaporated in vacuo and the remaining oil was chromatographed yielding58 (6678 mg, 25.49 mmol, 86%) as a viscous oil, which crystalized onstanding. This was recrystallized (hexane ether) giving a white solidmp. 61°-63° C.; IR (KBr): 3290, 2930, 1590, 1460, 1425, 1210, 1140, y1120 cm⁻¹ ; ¹ H NMR: 1.56 (m, 4H), 1.93 (m, 4H), 2.62 (bs, 1H, exc),3.26 (bs, 2H), 3.79 (s, 6H), 4.59 (s,2H), 5.20 (bs, 1H), and 6.54 (s,1H); ¹³ C NMR: 22.5, 23.1, 25.3, 28.8, 30.6, 55.8, 65.7, 102.5, 116.6,119.9, 136.3, 139-8, and 158.5; Elemental analysis: calcd. C=73.25,H=8.45; obsd. C=73.21, H=8.52.

Preparation of 59: A solution of n-butyllithium in hexane (4.2 ml, 8.4mmol) was added to compound 58 (1000 rag, 3.82 mmol) in hexane (50 ml)and TMEDA (0.69 ml, 4.58 mmol). After stirring at 0° C. for 15 rain andat room temperature for 90 min, the resulting suspension was cooled to-78° C. and dry carbon dioxide was bubbled for 1 hr at -78° C. andduring 1 hr at room temperature. After adding 2N NaOH (25 ml), theunreacted material was extracted with ether and the extracts werediscarded- The aqueous phase was acidified with 6N HC1 and the reactionproducts were extraced with ether (4×50 ml)- After drying (MgSO⁴ andconcentration in vacuo of the organic phase, 59 (727 mg, 2.52 mmol, 66%)was recovered as a solid mp. 103°-104.5° C. (recrystallized fromhexane-ether); IR (KBr): 3000-2820, 1740, 1600, 1460, 1420, 1340, 1240,1200, 1090, 1015, and 940 cm⁻¹ ; ¹ H NMR: 1.48-2.00 (m, 8H), 3.30 (s,2H), 3.88 (s, 3H), 4.03 (s, 3H), 5.19 (bs, 1H), 5.19 (s, 2H), and 6.64(s, 1H); ¹³ C NMR: 22.5, 23.1, 25.3, 28.9, 31.0, 56.2, 62.6, 68.8,98.7,109.8, 121.0, 123.0, 136.2, 148.8, 158.4 and 64.3; Elementalanalysis: calcd. C=70.83, H=6.94; C=70.77, H=6.97.

Preparation of 60: A solution of 59 (470 mg, 1.63 mmol) was demethylateduntil no more starting material was left, according to the TLC. Thereaction products were extracted with ethyl acetate (4×40 ml), thendried (MgSO4), concentrated in vacuo and purified. to yield 60 (332 mg,1.21 mmol, 74%) as a white solid mp. 150°-151° C. (recrystallized fromhexane-ether); IR (KBr): 3420, 3000-2800, 1720, 1600, 1465, 1345, 1280,1250, 1195 and 1075; ¹ H NMR: 1.45-2.00 (m, 8H), 3.27 (s, 2H), 3.88 (s,3H), 5.2 (s, 2H), 6.5 (s, 1H) and 7.71 (s, 1H); ¹³ C NMR: 2.4, 23.0,25.2, 28.7, 30.2, 56.2, 70.4, 96.1, 104.2, 15.5, 120.9, 135.4, 146.2,155.0, 165.2 and 172.8.

Preparation of 61: Amberlyst 15 (290 mg, 2 g/mmol) was added to asolution of 60 (40 mg, 0.146 mmol) in dry methylene chloride (2.9 ml, 20ml/mmol) after stirring overnight at room temperature, the solvent wasfiltered through a short column of silica gel, the solids were washedwith ethyl acetate and the filtrate was filtered through silica gel. Thecombined filtrates were concentrated in vacuo and chromatographed,affording 61 (38 mg, 0.139 mmol, 95%) as a solid mp. 132°-134° C.(recrystallized from hexane-ether); IR (KBr): 2980-2820, 1740, 1610,1440, 1330, 1280, 1260, 1235, 1205, 1145, 1080, 1010, 930 and 780 cm⁻¹,¹ H NMR: 1.35-2.00 (m, 10H), 2.89 (s, 2H), 3.91 (s, 3H), 5.20 (s, 2H)and 6.46 (s, 1H); ]3C NMR: 23.1, 25.0, 37.1, 55.9, 69.8, 93.7, 96.0,102.0, 114.8, 150.2, 157.8, 162.1 and 169.6; Elemental analysis: calcd.C=70.04, H=6.62; obsd. C=70.11, H=6.62.

Preparation of 62: Compound 61 (211 mg, 0.77 mmol) was dissolved in THF(4 ml) and the resulting solution was added to a solution of 10N NaOH (6ml, 60 mmol) and FTBA (1 ml, 1 mmol). Potassium permanganate was (KMnO₄)added to the system until no more starting material was observed by TLC.The excess of KMnO₄ was destroyed with sodium sulfite and the reactionwas acidified with 6N H₂ SO₄. The organic material was extracted withethyl acetate (4×50 ml), the extracts were dried (MgSO₄), concentratedin vacuo and chromatographed yielding 62 (120 mg, 0.41 mmol, 53%) as awhite solid mp. 128°-130° C. (recrystallized from hexane-ether); IR(KBr): 2950, 2850, 1830, 1770, 1625, 1455, 1340, 1270, 1210, 1145, 990,985 and 745 cm ⁻¹ ; ¹ H NMR: 1.35-1.95 (m, 10H), 2.86 (s, 2 H), 3.91 (s,3H), 5.90 (bs, 1H), 6.53 (s,lH) and 6.61 (s, 1H); ¹³ C NMR: 23.1, 24.9,37.0, 37.1, 56.0, 93.9, 97.3, 98.0, 102.6, 116.4, 150.1, 157.3, and162.1 and 167.6; Elemental analysis: calcd. C=66.20, H=6.25; obsd.C=66.03, H=6.18.

8.2. SYNTHESIS OF 6-CARBOXY-7-FORMYL-4-METHOXY-SPIRO[BENZOFURAN-2 (3H)-CYCLOHEXANE (68) AND 6,7-DICARBOXYL-4-METHOXYSPIRO [BENZOFURAN-2 (3H)-CYCLOHEXANE] (66)

Preparation of 65: A solution of n-butyllithium in hexane (2.20 ml, 4.95mmol) was added to a solution of N, N, N' trimethylethylenediamine (0.65ml, 5.09 mmol) in THF (6 ml) at -20° C. (CCl₄ /CO₂). After 30 rain, 11b(1170 mg, 4.76 mmol, prepared as described in section 6.8 supra) in THF(4 ml) was added dropwise, followed 30 min later by n-BuLi (6.35 ml,14.28 mmol). The resulting system was kept at -20° C. for 24 h, and DMFwas added (02.20 ml, 28.56 mmol). After 24 h reaction period, thereaction products were partitioned between ether (4×50 ml) and brine (50ml), chromatography of the extracts gave 65 (1135 mg, 4.14 mmol, 87%) asa solid mp. 129°-131° C. (recrystallized from hexane-ether); IR (KBr):3000-2840, 1670, 1600, 1470, 1425, 1390, 1320, 1280, 1260, 1210, 1130,1030, 890, 850, 770, 700 and 620 cm⁻¹ ; ¹ H NMR: 1.40-1.93 (s, 10H),2.93 (s, 2H), 3.94 (s, 3H) 7.04 (s, 1H), 10.35 (s, 1H) and 10.70 (s,1H); ¹³ C NMR: 22.9, 24.9, 37.2, 37.7, 56.0, 93.0, 103.6, 113.7, 120.2,138.7, 160.3, 164.7, 188.6 and 192.6; Elemental analysis: calcd.C=70.06, H=6.61; obsd. C=69.84, H=6.65.

Preparation of 68: A 4N solution of potassium hydroxide (52.92 mmol) wasadded dropwise to a stirred solution of compound 65 (2900 mg, 1058 mmol)in THF (10 ml) and water (7 ml) containing dissolved silver nitrate(3777 mg, 22.22 mmol, 1.05 eq. ) at room temperature. The reactionsystem was protected from direct light. After stirring for 2 h at roomtemperature, the solids were filtered and extensively washed withdistilled water. 2N H₂ SO₄ was added until pH 3, and the reactionproducts were extracted with ether (3×150 ml), washed with brine (2×25ml) and dried (MgSO₄). Chromatography of the reaction products allowedthe recovery of the starting material (1823 mg, 63%), compound 68 (326mg, 1.12 mmol, 11%, 29% corrected yield) and compound 66 (614 mg, 2.01mmol, 19%, 51%). Compound 68: mp: 148°-150° C. (rec. from hexane-ether);IR (KBr): 3440, 3000-2840, 1740, 1630, 1450, 1345, 1290, 1270, 1150,1105, 1010, 910, 860, 770, 690, cm⁻¹ ; ¹ H NMR (DMSO d₆): 1.30-1.52 (m,4H), 1.62-1.81 (m, 6H), 2.91 (s, 2H), 3.87 (s, 3H), 6.55 (bs, 1H), 6.85(s, 1H), and 7.95 (bs, 1H); ¹³ C NMR (DMSO d₆): 22.52, 24.47, 36.57,37.56, 55.86, 91.90, 96.33, 98.34, 120.60, 121.48, 128.99, 154.0-3,158.28, 168.42; MS (e/m, %): 290 (M+, 89), 289 (72), 272 (100), 271(88), 244 (65), 215 (78), 192 (98), 191 (95), 190 (65), 165 (93), 164(82), 79 (89). Elemental analysis: calcd. C=66.20, H=6.25; obsd.C=66.19, H=6.26.

Direct Preparation of 66: To a solution 65 (153 mg, 0.56 mmol) inethanol (5 ml), was added a solution of silver nitrate (222 mg, 1.30mmol) in distilled water (1 ml), followed by KOH (3 ml, 2.99 mmol). Thesystem was stirred overnight at room temperature, shielded from thelight, then it was filtered and the residue was carefully washed withwater. The combined aqueous phases were extracted with ether, theaqueous phase was then acidified and extracted with ether (3×25 ml); thecombined organic phases were dried and chromatographed to afford 66 (156mg, 0.51 mmol, 91%) as a white solid mp. 188°-190° C. (recrystallizedfrom acetone); IR (KBr): 3500-2400, 3000-2850, 1700, 1610, 1410, 1330,1290, 1130, 1040, 1000, 930, 855, 750 and 660 cm⁻ : ¹ H NMR: (acetoned₆): 1.40-1.90 (m, 10H), 2.93 (s, 2H), 3.91 (s, 3H) and 6.95 (s, 2H); ¹³C NMR: (acetone d₆): 23.5, 25.6, 37.6, 38.7, 56.1, 91.7, 105.1, 111.6,118.8, 133.0, 157.8, 158.8, 167.2 and 186.1; Elemental analysis: calcd.C=62.74, H=5.92; obsd. C=62.66, H=6.01.

8.3. SYNTHESIS OF 4-METHOXY-5a, 6,8,8a-TETRAHYDROSPIRO [BENZO[2,1-b: 3,4-C']DIFURAN-2(8H) CYCLOHEXAN]-6,8-DIONE;6,7-DIHYDROXYMETHYL-4-METHOXYSPIRO [BENZOFURAN-2(3H) CYCLOHEXANE]; AND4-METHOXYSPIRO[BENZO[2, 1-B: 3,4-C']DIFURAN-2 (3H) -CYCLOHEXAN]-6(8H)-ONE

Preparation of 63: A cold and stirred solution of 62(150 mg, 0.52 mmol)in acetone (10 ml) was treated with excess of Jones reagent (Bowden etal., 1946, J. Chem. Soc. 39; Bowers et al., 1953, J. Chem. Soc., 2548)until the starting material was completely converted (TLC). The excessreagent was destroyed with isopropanol, brine was added (30 ml) and thereaction products were extracted with ethyl acetate (4×50 ml) theextracts were dried (MgSO₄), concentrated in vacuo and chromatograhphedyielding 63 (134 mg, 0.46 mmol, 89%) as a white solid mp. 197°-198.5° C.(recrystallized from acetone); IR (KBr): 3400, 2940, 2860, 1740, 1620,1450, 1330, 1235, 1140, 1090, 1020, 930 and 760 cm⁻¹ ; ¹ H NMR (acetoned₆): 1.45-1.95 (m, 10H), 3.03 (s, 2H), 4.08 (s, 3H), 7.15 (s, 1tt); ¹³ CNMR (acetone d₆): 23.5, 25.5, 37.7, 38.3, 57.2, 95.6, 102.1, 123.9,134.9, 158.4, 161.2, 163.8 and 164.2; MS (m/z, %): 288 (M+, 40), 287(32), 231 (40), 208 (49), 207 (52), 85 (27), 81 (100), 77 (36), 71 (28),69 (42), 57 (67) and 55 (61); HRMS: expected 288.0998 for C₁₆ H₁₆ O₅,obsd.: 288.0999.

Preparation of 64: A solution of 61 (300 mg, 1.09 mmol) in THF (15 ml)was treated for 3 h with LAH (slight excess). After adding Na₂ SO₄.10H₂O and ether, the reaction products were filtered, affording the diol 64(260 mg, 0.94 mmol, 86%); ¹ H NMR: 1.40-1.90 (m, 10H), 2.93 (s, 2H),3.83 (s, 3H), 4.66 (s, 2H), 4.70 (s, 2H) and 6.47 (s, 1H).

Preparation of 67: To a solution of 64 (260 mg, 0.935 mmol) in drymethylene chloride (15 ml) BaMnO₄ (1197 mg, 4.68 mmol) was added and theresulting suspension was stirred for 3 days at room temperature. Afterrecovering the reaction products by partition between brine (30 ml) andethyl acetate (3×50 ml) it was observed that this was an equimolecularmixture of 67 and 65.

9. COMPLEMENT INHIBITION BY DI- AND TRI- SUBSTITUTEDSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANES]

The 4 substituted spirobenzofuran compounds of example 7 and thedisubstituted spirobenzofuran compounds of example 8 were tested fortheir capacity to inhibit complement-mediated lysis of sheep red bloodcells (SRBC) as described in Section 6.6.2 supra with the modificationthat the human serum used as the complement source was diluted to 1 to100 in Hepes buffer, instead of 1 to 125. The results of the hemolysisassays are shown in FIG. 8 and Tables XI and XII.

                  TABLE XI                                                        ______________________________________                                        Inhibition of Hemolysis by 6,7-disubstituted                                  4-methoxy spiro[benzofuran-2(3H)-cyclohexanes]                                              Mean IH.sub.50 (± SD)*                                       Compound      mM            n=                                                ______________________________________                                        11a           1.330 (± 0.490)                                                                          10                                                44a           0.530 (± 0.190)                                                                          23                                                62            1.670 (± 0.153)                                                                          3                                                 66            0.800 (± 0.356)                                                                          3                                                 68            0.164 (± 0.076)                                                                          7                                                 K76COOH       0.570 (± 0.170)                                                                          9                                                 ______________________________________                                         *The concentration of compound (± standard deviation) required to          produce a value for hemolysis inhibition of 0.5 as described in Section       6.36.2 supra.                                                            

                  TABLE XII                                                       ______________________________________                                        Inhibition of Hemolysis by 6-carboxyl-4-substituted                           spiro[benzofuran-2(3H)-cyclohexanes]                                                        Mean IH.sub.50 (± SD)*                                       Compound      mM            n=                                                ______________________________________                                        31a           1.45 (±0.21)                                                                             2                                                 44a           0.532 (±0.193)                                                                           29                                                44b           0.580 (±0.216)                                                                           3                                                 55a           2.53 (±1.00)                                                                             2                                                 55b           0.430         1                                                 55C           0.280 (±0.014)                                                                           2                                                 55d           >2.8          1                                                 55e           0.305 (±0.049)                                                                           2                                                 55g           2.320 (±0.099)                                                                           2                                                  55h**        0.320 (±0.056)                                                                           2                                                 55i           1.45 (±0.44)                                                                             2                                                 K76COOH       0.570 (±0.170)                                                                           9                                                 ______________________________________                                         *The concentration of compound (± standard deviation) required to          produce a value for hemolysis inhibition of 0.5 as described in Section       6.36.2 supra.                                                                 **The sodium salt of                                                          4p-aminophenoxy-carboxyspiro[benzofuran2(3H)-cyclohexane                 

By comparing the inhibition of hemolysis by compounds 62, 66 and 68 asshown in FIG. 8 and Table XI, it can be seen that the particulararrangement of substitutents at positions 6 and 7 of the benzofuran ringis important for anti-hemolytic activity. By comparing IH₅₀ values forthe position 4-substituted series as shown in Table XII (for example,the values for 55c, 55e, and 55h), improvements in anti-hemolyticactivity can also be obtained through the optimal choice of the Rsubstituent at position 4. In addition, several of the compounds (seefor example, compounds 55c, 55e, 55h and 68 in particular) are moreeffective in inhibiting complement-mediated hemolysis than is K76COOH.It is anticipated that optimal substituents at position 4, combined withan optimal pair of substituents at position 6 and 7, will result in evenmore potent complement inhibitors.

10. IN VITRO AND IN VIVO INHIBITION OF COMPLEMENT BY DI- ANDTRI-SUBSTITUTED SPIRO[BENZOFURAN-2 (3H) -CYCLOHEXANES] 10.1.INTRODUCTION

The role of complement in hyperacute graft rejection has beendemonstrated in animal models of both sensitized allograft (Knechtle etal., J. Heart Transplant., 1983, 4:541; Pruitt and Bollinger, J. Surg.Rest, 1991, 50:350-355; as well as xenograft transplantation (Adachi etal., Transplant Proc., 1987, 119: 1145-1148; Migagawa et al.,Transplantation, 1988, 46:825; Prewitt et al., 1991, Transplantation52:868-873). We have chosen an established discordant xenograft model, aguinea pig-to-rat cardiac transplant, to assess the effects of thesubstituted dihydrobenzofurans on hyperacute rejection in vivo.

10.2. INHIBITION IN VITRO OF RAT AND HUMAN COMPLEMENT

Compounds 68 and 44a were prepared and characterized as described insections 8.2 and 7.1, respectively. These compounds were demonstrated toinhibit complement-mediated hemolysis using human serum as a complementsource, as described in section 6.6.2 and Section 9, Supra. Compounds 68and 44a were also demonstrated to inhibit rat complement using themethods of section 6.6.2, except that rat serum was substituted forhuman serum as the complement source. A soluble human recombinantcomplement receptor type 1 (sCR1) was prepared as described (Weisman etal., Science, 1990, 249:146-151; Yeh et al., J. Immunol, 1991; andInternational Patent Publications No. WO 89/09220 and WO 9/05047, bothentitled "The human C3b/C4b receptor (CR1)" Fearon et al.) and used as apositive control the in vitro assays. The results of these studies aresummarized in Table XIII.

                  TABLE XIII                                                      ______________________________________                                        Inhibition of rat or human complement-mediated                                hemolysis by substituted spiro[benzofuran-2(3H)-                              cyclohexanes] and by sCR1                                                                 IH.sub.50 * for Human                                                                      IH.sub.50 for Rat                                    Compound    Complement   Complement                                           ______________________________________                                        sCR1        0.9 nM         1.5 nM                                             68          100 μM      2500 μM                                                     164 μM    11,000 μM                                         44a         538 μM    17,300 μM                                         ______________________________________                                         *The concentration of compound required to produce a value for hemolysis      inhibition of 0.5 as described in Section 6.6.2, supra.                  

As seen in Table XIII, the human sCR1 protein inhibits rat and humancomplement with similar potency in this assay. The compounds, on theother hand, more effectively inhibit human than rat complement.Twenty-five- to 70-fold higher concentrations of compound 68 wererequired to inhibit rat complement-mediated lysis than were required toinhibit human complement-mediated lysis to similar levels.

10.3.INHIBITION OF ALTERNATIVE PATHWAY-MEDIATED HEMOLYSIS

The inhibition of sheep red blood cell lysis as described in section6.6.2 predominantly reflects on classical complement pathway activationsince it is initiated by the antigen-antibody complexes on the surfaceof the sensitized erythrocytes. The assay probably includes a minorcontribution from alternative pathway activation as well.

The capacity of the substituted dihydrobenzofurans to inhibit thealternative complement pathway was assessed using the modified method ofPlatts-Mills and Ishizaka, 1974, J. Immunol., 113: 348-358. Essentially,rabbit erythrocytes were lysed using human serum as complement ingelatin veronal buffer (GVB, Sigma, St. Louis, Mo.) with added EGTA(ethylene glycol-bis[β-aminoethyl ether]N, N, N^(l),N¹ -tetraaceticacid) and Mg²⁺ to 8 mM and 2 mM, respectively. Rabbit erythrocytes(4.5×10⁶ cells/ml), human serum (1 in 24 dilution), and the compounds tobe tested were incubated one hour at 37° C. in a V-bottom microtiterplate, cells pelleted by centrifugation, the supernatants transferred toa flat-bottom microtiter plate, and the absorbance at 410 nm determined.Samples were paired with identical controls lacking human serum(complement-independent lysis). Both samples and controls were run intriplicate. Control values were subtracted from sample values and thefractional inhibition was determined relative to the uninhibited (noadded compound) sample. Results are reported as the concentration ofcompound yielding fifty per cent (50%) inhibition, AH₅₀. The AH₅₀ valuesfor several of the compounds are included along with the results ofother in vitro assays in Table XIV. As seen in Table XIV, compound 68inhibited the classical pathway more effectively than K76COOH, but wassomewhat less potent than K76COOH in inhibiting alternative complementpathway. Compound 44a inhibited the classical pathways as well asK76COOH, but was clearly less potent than K76COOH or 68 in inhibitingthe alternative pathway.

10.4. INHIBITION OF C3b PROTEOLYSIS BY FACTOR I

K76COOH has been shown to inhibit the proteolysis of C3b by factor I andthe cofactor protein H (Hong et al., 1981, J. Immunol., 127:104-108. Thecapacity of the substituted dihydrobenzofurans to inhibit the specificdegradation of the C3b α-chain by factor I was assessed on sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Wong etal., 1985, J. Immunol. Methods, 82:303-313; Weisman et al., 1990,Science, 249:146-151) using either factor H or sCR1 as cofactor.Purified C3b at 1.2 μM, factor I (Cytotech, San Diego, Calif.) at 0.1μM, sCR1 at 8 nM as a cofactor, and test compound at varyingconcentrations, were incubated for 60 minutes at 37° C. The reactionswere stopped by the addition of reducing SDS-PAGE sample buffer(Laemmli, 1970, Nature, 227:680-685) subjected to electrophoresis on4-20% polyacrylamide gradient gels, visualized with Coomassie Bluestain, and quantitated by densitometry. Controls lacking factor I andcontrols with factor I but lacking test compounds were included. Resultsare reported as the concentration of test compound which inhibitedapproximately fifty percent (50%) of the C3b α-chain degradation, ICs₅₀.

The results for several compounds are included in Table XIV.

                  TABLE XIV                                                       ______________________________________                                        In vitro assays of complement inhibition by                                   substituted spiro[benzofuran-2(3H)-cyclohexanes].                             Concentration* (μM) yielding 50% inhibition.                                       Classical   Alternative  C3b clevage                                          pathway IH.sub.50                                                                         pathway AH.sub.50                                                                          by Factor I                                  Compound                                                                              (μM)     (μM)      (μM)                                      ______________________________________                                        K76COOH 570 (±170;                                                                             850 (n = 1)  110                                                  n = 9)                                                                68      160 (±69;                                                                              1360 (±320;                                                                             150                                                  n = 9)      n = 3)                                                    44a     530 (±190;                                                                             >5030        2740                                                 n = 23                                                                ______________________________________                                         *Values reported are the mean (± standard deviation; sample number, n)     Compound 68 and K76COOH were equally effective in inhibiting proteolysis      of C3b by factor I using sCR1 as a cofactor.                             

10.5. GUINEA PIG-TO-RAT CARDIAC TRANSPLANT

The effects of compounds 68 and 44a on hyperacute graft rejection weretested in a model of discordant xenografting. The methods were the sameas described previously (Prewitt et al., 1991, Transplantation52:868-873) except that the test compounds were substituted for theactive agent (sCR1) of the earlier study. As indicated in the earlierstudy, male Lewis rats, 12-16 weeks of age, and male Hartley guineapigs, 4-10 weeks of age (Charles River, Wilmington, Mass.) served ascardiac xenograft recipients and donors, respectively. Under Halothaneanesthesia, heterotopic cardiac xenotransplantation was performed by amodification of the microvascular technique of Ono and Lindsey, 1990, J.Thorac. Cardiovas. Surg., 57:225, with anastomosis of the donor aorta tothe recipient infrarenal aorta and the donor pulmonary artery to therecipient inferior vena cava. Cardiac xenografts were evaluated visuallyover the first 30 minutes following reperfusion and then, followingabdominal closure, every 10 minutes by palpation until rejection.Rejection was defined, as total cessation of cardiac xenograftcontraction and was confirmed by direct visualization and histologicalexamination. The compounds 68 and 44a were prepared as 14 mg/mlsolutions in phosphate buffered saline (PBS) and were administered as a2.0 ml intravenous bolus into the recipient inferior vena cava, superiorto the caval cross clamp, immediately prior to cardiac xenograftreperfusion. Control animals received similar volumes of PBS. Three(n=3) control animals were tested concurrent with the animals receivingcompound 68; data from historic controls (n=13) subjected to the sameprocedure and previously reported (Pruitt et al., 1991, Transplantation52:868-873) are also included for comparison. Table XV includes thexenograft survival times (minutes) for individual animals and the meansand standard errors of the mean (SEM) for the various groups. As can beseen in the table, compound 68 treatment resulted in prolonged graftsurvival times in 2 of the 3 animals tested relative to controls.

                  TABLE XV                                                        ______________________________________                                        Guinea pig-to-rat cardiac transplant                                                      Graft survival  Mean ± SEM                                     Treatment   time (minutes)  minutes)                                          ______________________________________                                        Historic con-                                                                             5, 7, 7, 9, 9, 10, 10,                                                                        17 ± 4                                         trols (PBS) 11, 16, 17, 24, 40, 60                                            Concurrent con-                                                                           5, 7, 19        10 ± 4                                         trols (PBS)                                                                   68 at 28 mg/rat                                                                           7, 150, 270     142 ± 76                                       44a at 28 mg/rat                                                                          7, 9            8                                                 ______________________________________                                    

The present invention is not to be limited in scope by the foregoingexamples, which are intended as purely illustrative of specific aspectsof the invention and any compounds that are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference herein in their entireties.

What is claimed is:
 1. A compound of the general formula, 4: ##STR19##in which R represents a hydrogen atom, a lower alkyl group, asubstituted lower alkyl group, a benzyl group, a substituted benzylgroup, a phenyl group or a substituted phenyl group; R₁ and R₂ representindependently, a carboxylic acid group, a formyl group, a hydroxymethylgroup, a N-(lower alkyl)carbamoyl group, a trifluoroacetyl group, ahalide group, a vinyl group, a substituted vinyl group having up to 10carbon atoms, an alkylidene group having up to 20 carbon atoms, analiphatic acyl group, a substituted aliphatic acyl group, an aromaticacyl group, a substituted aromatic acyl group, a sulfamoyl group, anaminomethyl group, a N-(lower alkyl)aminomethyl group, a N,N-di(loweralkyl)aminomethyl group, a heterocyclic ring, an N-(acyl)carbamoylgroup, an amidino group or a hydrazide group; R_(l) and R₂ together withthe carbon atoms to which they are attached may also represent a cyclicanhydride or lactone; or a pharmaceutically acceptable acid or baseaddition salt or ester thereof.
 2. The compound of claim 1 which isselected from the group consisting of 6,7-dicarboxy-4-methoxyspiro[benzofuran-2 (3H) -cyclohexane],6,7-diformyl-4-methoxyspiro[benzofuran-2(3H) -cyclohexane],6,7-di(hydroxymethyl)-4-methoxyspiro(benzofuran-2(3H)-cylcohexane],4-methoxy-3,6-dihydrospiro[benzo[2,1-b:3,4-c']difuran-2(SH)-cyclohexane]-8-one,7-carboxy-6-formyl-4methoxyspiro [benzofuran-2(3H)-cyclohexane],4methoxy, 5a,6,8,8a-tetrahydrospiro[benzo[2,1-b:3,4-c']difuran-2(8H)-cyclohexan]-6,8-dione,and6-carboxy-7-hydroxymethyl-4-methoxyspiro[benzofuran-2(3H)cyclohexane]lactone.3. The compound of claim 1 which is selected from the group consistingof 6-carboxy-7-formyl-4-methoxyspiro[brenzofuran-2(3H)-cyclohexane] and6-carboxy-7-formyl-4-phenoxyspiro[benzofuran-2(3H)-cyclohexane].
 4. Thecompound of the general formula, 4: ##STR20## in which R represents ahydrogen atom, a lower alkyl group, a substituted lower alkyl group, abenzyl group, a substituted benzyl group, a phenyl group or asubstituted phenyl group; R₁ represents a carboxylic acid group, or abioisosteric acid or base group; R₂ represents a formyl group or abioisosteric neutral group; or a pharmaceutically acceptable acid orbase addition salt or ester thereof.
 5. The compound of claim 4 in whichsaid bioisosteric base group is a carbamoyl group, a sulfamoyl group, anN-acylcarbamoyl group or a tetrazole ring.
 6. The compound of claim 4 inwhich said bioisosteric base is an aminomethyl group, a N-(loweralkyl)aminomethyl group, a N,N-di(lower alkly)aminomethyl group, anoxazoline ring, an amidino group or a hydrazide group.
 7. The compoundof claim 4 in which said bioisosteric neutral group is an aliphatic acylgroup, a substituted aliphyatic acyl group, an aromatic acyl group, or asubstituted aromatic acyl group.
 8. A compound of the general formula,4: ##STR21## in which R represents a hydrogen atom, a lower alkyl group,a substituted lower alkyl group, a benzyl group, a substituted benzylgroup, a phenyl group or a substituted phenyl group; R_(l) and R₂represent independently a hydrogen atom, a carboxylic acid group, aformyl group, a hydroxymethyl group, a N-(lower alkyl)carbamoyl group, atrifluoroacetyl group, a halide group, a vinyl group, a substitutedvinyl group having up to 10 carbon atoms, an alkylidene group having upto 20 carbon atoms, an aliphatic acyl group, a substituted aliphaticacyl group, an aromatic acyl group, a substituted aromatic acyl group, asulfamoyl group, an aminomethyl group, a N-(lower alkyl)aminomethylgroup, a N,N-di(lower alkyl)aminomethyl group, a heterocyclic ring, anN-(acyl)carbamoyl group, an amidino group or a hydrazide group; R_(l)and R₂ together with the carbon atoms to which they are attached mayalso represent a cyclic anhydride or lactone; or a pharmaceuticallyacceptable acid or base addition salt or ester thereof, provided that R₁and R₂ do not both represent hydrogen atoms.
 9. A pharmaceuticalcomposition for use in a method of treating a patient with an immunedisorder or a disorder involving undesirable or inappropriate complementactivity comprising an effective amount of the compound of claim 1, 3, 4or
 8. 10. The composition of claim 9 in which the immune disorder ordisorder involving undesirable or inappropriate complement activity isselected from the group consisting of soft tissue destruction due toburn, myocardial infarct induced trauma, adult respiratory distresssyndrome, and myocardial ischmia and reperfusion; specific andnon-specific proteolytic processing of C5; inflammation associated withkidney stones, systemic lupus erythematosis, nephrotoxicglomeronephritis, and multiple sclerosis; atrophic gastritus,thyroiditis, allergic encephalomyelitis, gastric mucosa, thyrotoxicosis,autoimmune hemolytic anemia, pemphigus vulgaris, sympathetic opthalmia,delayed-type hypersensitivity, autoimmune disorders and drug allergies;and tissue plasminogen activator therapy and cardiopulmonary bypass. 11.A pharmaceutical composition for use in a method of treating a patientwith graft rejection, graft-host reaction, or organ transplant rejectioncomprising an effective amount of the compound of claim 1, 3, 4 or 8.12. The composition of claim 8 in which the graft is a xenograft or anallograft.
 13. The composition of claim 8 in which the graft is a hearttransplant.